IL-1 zeta, IL-1 zeta splice variants and Xrec2 DNAS and polypeptides

ABSTRACT

The invention is directed to novel, purified and isolated IL-1 zeta and Xrec2 polypeptides and fragments thereof, the nucleic acids encoding such polypeptides, processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides, fragmented peptides derived from these polypeptides, and uses thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of InternationalApplication PCT/US99/29549, with an international filing date of Dec.14, 1999, and claims the benefit of U.S. Provisional Application No.60/164,675, filed on Nov. 10, 1999, and U.S. Provisional Application No.60/112,163, filed on Dec. 14, 1998.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention is directed to novel, purified and isolated IL-1zeta, IL-1 zeta splice variants and Xrec2 polypeptides and fragmentsthereof, the nucleic acids encoding such polypeptides, processes forproduction of recombinant forms of such polypeptides, antibodiesgenerated against these polypeptides, fragmented peptides derived fromthese polypeptides, and uses thereof.

[0004] 2. Description of Related Art

[0005] Interleukin-1 (IL-1) is a member of a large group of cytokineswhose primary function is to mediate immune and inflammatory responses.There are five known IL-1 family members which include IL-1 alpha(IL-1α), IL-1 beta (IL-1β), IL-1 receptor antagonist (IL-1ra), IL-1delta (IL-1δ) as disclosed in US/99/00514), and IL-18 (previously knownas IGIF and sometimes IL-1 gamma). IL-1 that is secreted by macrophagesis actually a mixture of mostly IL-1β and some IL-1α (Abbas et al.,1994). IL-1α and IL-1β, which are first produced as 33 kD precursorsthat lack a signal sequence, are further processed by proteolyticcleavage to produce secreted active forms, each about 17 kD.Additionally, the 33 kD precursor of IL-1α is also active. Both forms ofIL-1 are the products of two different genes located on chromosome 2.Although the two forms are less than 30 percent homologous to eachother, they both bind to the same receptors and have similar activities.

[0006] IL-1ra, a biologically inactive form of IL-1, is structurallyhomologous to IL-1 and binds to the same receptors. Additionally, IL-1rais produced with a signal sequence which allows for efficient secretioninto the extracellular region where it competitively competes with IL-1(Abbas et al., 1994).

[0007] The IL-1 family of ligands binds to a family of two IL-1receptors, which are members of the Ig superfamily. IL-1 receptorsinclude the 80 kDa type I receptor (IL-1RI) and a 68 kDa type IIreceptor (IL-1RII). IL-1 ligands can also bind to a soluble proteolyticfragment of IL-1RII (sIL-1RII) (Colotta et al., 1993).

[0008] The major source of IL-1 is the activated macrophage ormononuclear phagocyte. Other cells that produce IL-1 include epithelialand endothelial cells (Abbas et al., 1994). IL-1 secretion frommacrophages occurs after the macrophage encounters and ingestsgram-negative bacteria. Such bacteria contain lipopolysaccharide (LPS)molecules, also known as endotoxin, in the bacterial cell wall. LPSmolecules are the active components that stimulate macrophages toproduce tumor necrosis factor (TNF) and IL-1. In this case, IL-1 isproduced in response to LPS and TNF production. At low concentrations,LPS stimulates macrophages and activates B-cells and other hostresponses needed to eliminate the bacterial infection; however, at highconcentrations, LPS can cause severe tissue damage, shock, and evendeath.

[0009] The biological functions of IL-1 include activating vascularendothelial cells and lymphocytes, local tissue destruction, and fever(Janeway et al., 1996). At low levels, IL-1 stimulates macrophages andvascular endothelial cells to produce IL-6, upregulates molecules on thesurface of vascular endothelial cells to increase leukocyte adhesion,and indirectly activates inflammatory leukocytes by stimulatingmononuclear phagocytes and other cells to produce certain chemokinesthat activate inflammatory leukocytes. Additionally, IL-1 is involved inother inflammatory responses such as induction of prostaglandins, nitricoxide synthetase, and metalloproteinases. These IL-1 functions arecrucial during low level microbial infections. However, if the microbialinfection escalates, IL-1 acts systemically by inducing fever,stimulating mononuclear phagocytes to produce IL-1 and IL-6, increasingthe production of serum proteins from hepatocytes, and activating thecoagulation system. Additionally, IL-1 does not cause hemorrhagicnecrosis of tumors, suppress bone marrow stem cell division, and IL-1 islethal to humans at high concentrations.

[0010] Given the important function of IL-1, there is a need to identifyadditional members of the IL-1 ligand family and the IL-1 receptorfamily. In addition, in view of the continuing interest in proteinresearch and the immune system, the discovery, identification, and rolesof new proteins and their inhibitors, are at the forefront of modernmolecular biology and biochemistry. Despite the growing body ofknowledge, there is still a need in the art to discover the identity andfunction of proteins involved in cellular and immune responses.

[0011] In another aspect, the identification of the primary structure,or sequence, of an unknown protein is the culmination of an arduousprocess of experimentation. In order to identify an unknown protein, theinvestigator can rely upon a comparison of the unknown protein to knownpeptides using a variety of techniques known to those skilled in theart. For instance, proteins are routinely analyzed using techniques suchas electrophoresis, sedimentation, chromatography, sequencing and massspectrometry.

[0012] In particular, comparison of an unknown protein to polypeptidesof known molecular weight allows a determination of the apparentmolecular weight of the unknown protein (T. D. Brock and M. T. Madigan,Biology of Microorganisms 76-77 (Prentice Hall, 6d ed. 1991)). Proteinmolecular weight standards are commercially available to assist in theestimation of molecular weights of unknown protein (New England BiolabsInc. Catalog:130-131, 1995; J. L. Hartley, U.S. Pat. No. 5,449,758).However, the molecular weight standards may not correspond closelyenough in size to the unknown protein to allow an accurate estimation ofapparent molecular weight. The difficulty in estimation of molecularweight is compounded in the case of proteins that are subjected tofragmentation by chemical or enzymatic means, modified bypost-translational modification or processing, and/or associated withother proteins in non-covalent complexes.

[0013] In addition, the unique nature of the composition of a proteinwith regard to its specific amino acid constituents results in uniquepositioning of cleavage sites within the protein. Specific fragmentationof a protein by chemical or enzymatic cleavage results in a unique“peptide fingerprint” (D. W. Cleveland et al., J. Biol. Chem.252:1102-1106, 1977; M. Brown et al., J. Gen. Virol. 50:309-316, 1980).Consequently, cleavage at specific sites results in reproduciblefragmentation of a given protein into peptides of precise molecularweights. Furthermore, these peptides possess unique chargecharacteristics that determine the isoelectric pH of the peptide. Theseunique characteristics can be exploited using a variety ofelectrophoretic and other techniques (T. D. Brock and M. T. Madigan,Biology of Microorganisms 76-77 (Prentice Hall, 6d ed. 1991)).

[0014] Fragmentation of proteins is further employed for amino acidcomposition analysis and protein sequencing (P. Matsudiara, J. Biol.Chem. 262:10035-10038, 1987; C. Eckerskom et al., Electrophoresis 1988,9:830-838, 1988), particularly the production of fragments from proteinswith a “blocked” N-terminus. In addition, fragmented proteins can beused for immunization, for affinity selection (R. A. Brown, U.S. Pat.No. 5,151,412), for determination of modification sites (e.g.phosphorylation), for generation of active biological compounds (T. D.Brock and M. T. Madigan, Biology of Microorganisms 300-301 (PrenticeHall, 6d ed. 1991)), and for differentiation of homologous proteins (M.Brown et al., J. Gen. Virol. 50:309-316, 1980).

[0015] In addition, when a peptide fingerprint of an unknown protein isobtained, it can be compared to a database of known proteins to assistin the identification of the unknown protein using mass spectrometry (W.J. Henzel et al., Proc. Natl. Acad. Sci. USA 90:5011-5015, 1993; D.Fenyo et al., Electrophoresis 19:998-1005, 1998). A variety of computersoftware programs to facilitate these comparisons are accessible via theInternet, such as Protein Prospector (Internet site:prospector.uscf.edu), MultiIdent (Internet site:www.expasy.ch/sprot/multiident.html), PeptideSearch (Internet site:www.mann.embl-heiedelberg.de...deSearch/FR_PeptideSearch Form.html), andProFound (Internet site:www.chait-sgi.rockefeller.edu/cgi-bin/prot-id-frag.html). These programsallow the user to specify the cleavage agent and the molecular weightsof the fragmented peptides within a designated tolerance. The programscompare these molecular weights to protein molecular weight informationstored in databases to assist in determining the identity of the unknownprotein. Accurate information concerning the number of fragmentedpeptides and the precise molecular weight of those peptides is requiredfor accurate identification. Therefore, increasing the accuracy indetermining the number of fragmented peptides and their molecular weightshould result in enhanced likelihood of success in the identification ofunknown proteins.

[0016] In addition, peptide digests of unknown proteins can be sequencedusing tandem mass spectrometry (MS/MS) and the resulting sequencesearched against databases (J. K. Eng, et al., J. Am. Soc. Mass Spec.5:976-989 (1994); M. Mann and M. Wilm, Anal. Chem. 66:4390-4399 (1994);J. A. Taylor and R. S. Johnson, Rapid Comm. Mass Spec. 11:1067-1075(1997)). Searching programs that can be used in this process exist onthe Internet, such as Lutefisk 97 (Internet site:www.lsbc.com:70/Lutefisk97.html), and the Protein Prospector, PeptideSearch and ProFound programs described above. Therefore, adding thesequence of a gene and its predicted protein sequence and peptidefragments to a sequence database can aid in the identification ofunknown proteins using tandem mass spectrometry.

[0017] Thus, there also exists a need in the art for polypeptidessuitable for use in peptide fragmentation studies, for use in molecularweight measurements, and for use in protein sequencing using tandem massspectrometry.

SUMMARY OF THE INVENTION

[0018] The present invention provides isolated nucleic acids andpolypeptides encoded by the nucleic acids for an IL-1 family ligandtermed “IL-1 zeta” and three splice variants of IL-1 zeta, termed TDZ.1,TDZ.2, and TDZ.3. The present invention also provides isolated nucleicacid molecules and polypeptides encoded by the nucleic acid moleculesfor an IL-1 family receptor termed “Xrec2.” Thus, in one aspect, theinvention is directed to isolated nucleic acid molecules of IL-1 zeta,TDZ.1, TDZ.2, and TDZ.3 comprising the DNA sequence of SEQ ID NO:1, SEQID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively, and nucleic acidmolecules complementary to SEQ ID NO:1, 5, 6, and 7. Similarly, theinvention is directed to isolated nucleic acid molecules of Xrec2comprising the nucleic acid molecule of SEQ ID NO:2 and nucleic acidmolecules complementary to SEQ ID NO:2.. In another aspect, theinvention is directed to isolated IL-1 zeta, TDZ.1, TDZ.2, and TDZ.3polypeptides having the amino acid sequences SEQ ID NO:3 SEQ ID NO:8,SEQ ID NO:9, and SEQ ID NO:10, respectively, and nucleic acid moleculesencoding the polypeptides of SEQ ID NO:3, 8, 9, and 10. Further includedin the invention are isolated Xrec2 polypeptides comprising the aminoacid sequence of SEQ ID NO:4 and nucleic acid molecules that encode thepolypeptide of SEQ ID NO:4

[0019] Both single-stranded and double-stranded RNA and DNA nucleic acidmolecules are encompassed by the invention, as well as nucleic acidmolecules that hybridize to a denatured, double-stranded DNA comprisingall or a portion of SEQ ID NOs:1, 2, 5, 6, and 7 and/or a DNA thatencodes the amino acid sequences set forth in SEQ ID NOs:3, 4, 8, 9, and10. Also encompassed are isolated nucleic acid molecules that arederived by in vitro mutagenesis of nucleic acid molecules comprisingsequences of SEQ ID NOs:1, 2, 5, 6, and 7 that are degenerate fromnucleic acid molecules comprising sequences of SEQ ID NOs:1, 2, 5, 6,and 7, and that are allelic variants of DNA of the invention. Theinvention also encompasses recombinant vectors that direct theexpression of these nucleic acid molecules and host cells transformed ortransfected with these vectors.

[0020] In addition, the invention encompasses methods of using thenucleic acids noted above to identify nucleic acids encoding proteinshaving activities associated with IL-1 family ligands and receptors.Thus, the IL-1 zeta nucleic acid molecules can be used to identify theIL-1 zeta receptor while the Xrec2 nucleic acid molecule can be used toidentify the Xrec2 ligand.

[0021] In addition, these nucleic acids can be used to identify thehuman chromosomes with which the nucleic acids are associated. Thus, theIL-1 zeta, TDZ.1, TDZ.2, and TDZ.3 nucleic acids can be used to identifyhuman chromosome 2 while the Xrec2 nucleic acids can be used to identifyhuman chromosome X. Accordingly, these nucleic acids can also be used tomap genes on human chromosomes 2 and X, respectively; to identify genesassociated with certain diseases, syndromes, or other human conditionsassociated with human chromosomes 2 and X, respectively; and to studycell signal transduction and the immune system.

[0022] The invention also encompasses the use of sense or antisenseoligonucleotides from the nucleic acids of SEQ ID NOs:1, 2, 5, 6, and 7to inhibit the expression of the respective polynucleotide encoded bythe genes of the invention.

[0023] The invention also encompasses isolated polypeptides andfragments of IL-1 zeta and Xrec2 as encoded by these nucleic acidmolecules, including soluble polypeptide portions of SEQ ID NOs:3 4, 8,9, and 10, respectively. The invention further encompasses methods forthe production of these polypeptides, including culturing a host cellunder conditions promoting expression and recovering the polypeptidefrom the culture medium. Especially, the expression of thesepolypeptides in bacteria, yeast, plant, insect, and animal cells isencompassed by the invention.

[0024] In general, the polypeptides of the invention can be used tostudy cellular processes such as immune regulation, cell proliferation,cell death, cell migration, cell-to-cell interaction, and inflammatoryresponses. In addition, these polypeptides can be used to identifyproteins associated with IL-1 zeta, TDZ.1, TDZ.2, and TDZ.3 ligands andwith Xrec2 receptors.

[0025] In addition, the invention includes assays utilizing thesepolypeptides to screen for potential inhibitors or enhancers of activityassociated with the polypeptides of this invention. The presentinvention also includes assays and screening methods for identifyinginhibitors or enhancers of activities associated with counter-structuremolecules of the polypeptides of this invention. Further, methods ofusing these polypeptides in the design of inhibitors (e.g., engineeredreceptors that act as inhibitors) thereof are also an aspect of theinvention.

[0026] The present invention further encompasses therapeutic methodsutilizing antagonist and/or agonists of the polypeptides of thisinvention and antagonists or agonists discovered in accordance with thescreening methods of this invention. For example, IL-1 zeta polypeptidesof the present invention enhance the secretion of IL-12 from isolatedprimary human monocytes. In view of IL-12 activity associated withstimulating and enhancing immune responses and IL-12 activity inpromoting Th1 mediated diseases, IL-1 zeta polypeptide agonists,together with IL-1 zeta antagonists are useful for treating disease ormedical conditions associated with immune system imbalances,particularly imbalances involving cell-mediated immune responses. Forexample, inhibitors or antagonists of IL-1 zeta polypeptides can be usedto treat disease associated with abnormal Th-1 immune responses,including the deleterious effects of inflammation. Agonists of IL-1 zetapolypeptide activity are useful in treating disease responsive to IL-12stimulation such as certain infectious diseases, including Leishmania,parasitic diseases and diseases preferentially inhibited by a Th1 immuneresponse. Additionally Il-1 zeta polypeptides upregulate TNF expressionand thus antagonists of IL-1 zeta polypeptides are useful in treatinginflammatory conditions including rheumatoid arthritis, SLE, myastheniagravis, insulin-dependent diabetes mellitus, thyroiditis, etc. anddiseases preferentially inhibited by a Th1 immune response.

[0027] The invention further provides a method for using thesepolypeptides as molecular weight markers that allow the estimation ofthe molecular weight of a protein or a fragmented protein, as well as amethod for the visualization of the molecular weight markers of theinvention thereof using electrophoresis. The invention furtherencompasses methods for using the polypeptides of the invention asmarkers for determining the isoelectric point of an unknown protein, aswell as controls for establishing the extent of fragmentation of aprotein.

[0028] Further encompassed by this invention are kits to aid in thesedeterminations.

[0029] Further encompassed by this invention is the use of the IL-1 zetaand Xrec2 nucleic acid sequences, predicted amino acid sequences of thepolypeptide or fragments thereof, or a combination of the predictedamino acid sequences of the polypeptide and fragments thereof for use insearching an electronic database to aid in the identification of samplenucleic acids and/or proteins.

[0030] Isolated polyclonal or monoclonal antibodies that bind to thesepolypeptides are also encompassed by the invention, in addition the useof these antibodies to aid in purifying the polypeptides of theinvention.

BRIEF DESCRIPTION OF THE FIGURE

[0031]FIG. 1 diagrams the genomic structure of the IL-1 zeta locus.

DETAILED DESCRIPTION OF THE INVENTION

[0032] The nucleic acid molecules encompassed in the invention includethe following nucleotide sequences: Name: TDZ.3    1 ATGTCCTTTGTGGGGGAGAA CTCAGGAGTG AAAATGGGCT CTGAGGACTG (SEQ ID NO:7)   51GGAAAAAGAT GAACCCCAGT GCTGCTTAGA AGAGATCTTC TTTGCATTAG  101 CCTCATCCTTGAGCTCAGCC TCTGCGGAGA AAGGAAGTCC GATTCTCCTG  151 GGGGTCTCTA AAGGGGAGTTTTGTCTCTAC TGTGACAAGG ATAAAGGACA  201 AAGTCATCCA TCCCTTCAGC TGAAGAAGGAGAAACTGATG AAGCTGGCTG  251 CCCAAAAGGA ATCAGCACGC CGGCCCTTCA TCTTTTATAGGGCTCAGGTG  301 GGCTCCTGGA ACATGCTGGA GTCGGCGGCT CACCCCGGAT GGTTCATCTG 351 CACCTCCTGC AATTGTAATG AGCCTGTTGG GGTGACAGAT AAATTTGAGA  401ACAGGAAACA CATTGAATTT TCATTTCAAC CAGTTTGCAA AGCTGAAATG  451 AGCCCCAGTGAGGTCAGCGA TTAG Name: IL-1 zeta    1 ATGTCAGGCT GTGATAGGAG GGAAACAGAAACCAAAGGAA AGAACAGCTT (SEQ ID NO:1)   51 TAAGAAGCGC TTAAGAGGTCCAAAGGTGAA GAACTTAAAC CCGAAGAAAT  101 TCAGCATTCA TGACCAGGAT CACAAAGTACTGGTCCTGGA CTCTGGGAAT  151 CTCATAGCAG TTCCAGATAA AAACTACATA CGCCCAGAGATCTTCTTTGC  201 ATTAGCCTCA TCCTTGAGCT CAGCCTCTGC GGAGAAAGGA AGTCCGATTC 251 TCCTGGGGGT CTCTAAAGGG GAGTTTTGTC TCTACTGTGA CAAGGATAAA  301GGACAAAGTC ATCCATCCCT TCAGCTGAAG AAGGAGAAAC TGATGAAGCT  351 GGCTGCCCAAAAGGAATCAG CACGCCGGCC CTTCATCTTT TATAGGGCTC  401 AGGTGGGCTC CTGGAACATGCTGGAGTCGG CGGCTCACCC CGGATGGTTC  451 ATCTGCACCT CCTGCAATTG TAATGAGCCTGTTGGGGTGA CAGATAAATT  501 TGAGAACAGG AAACACATTG AATTTTCATT TCAACCAGTTTGCAAAGCTG  551 AAATGAGCCC CAGTGAGGTC AGCGATTAG Name: Xrec2    1ATGAAAGCTC CGATTCCACA CTTGATTCTC TTATACGCTA CTTTTACTCA (SEQ ID NO:2)  51 GAGTTTGAAG GTTGTGACCA AAAGAGGCTC CGCCGATGGA TGCACTGACT  101GGTCTATCGA TATCAAGAAA TATCAAGTTT TGGTGGGAGA GCCTGTTCGA  151 ATCAAATGTGCACTCTTTTA TGGTTATATC AGAACAAATT ACTCCCTTGC  201 CCAAAGTGCT GGACTCAGTTTGATGTGGTA CAAAAGTTCT GGTCCTGGAG  251 ACTTTGAAGA GCCAATAGCC TTTGACGGAAGTAGAATGAG CAAAGAAGAA  301 GACTCCATTT GGTTCCGGCC AACATTGCTA CAGGACAGTGGTCTCTACGC  351 CTGTGTCATC AGAAACTCCA CTTACTGTAT GAAAGTATCC ATCTCACTGA 401 CAGTGGGTGA AAATGACACT GGACTCTGCT ATAATTCCAA GATGAAGTAT  451TTTGAAAAAG CTGAACTTAG CAAAAGCAAG GAAATTTCAT GCCGTGACAT  501 AGAGGATTTTCTACTGCCAA CCAGAGAACC TGAAATCCTT TGGTACAAGG  551 AATGCAGGAC AAAAACATGGAGGCCAAGTA TTGTATTCAA AAGAGATACT  601 CTGCTTATAA GAGAAGTCAG AGAAGATGACATTGGAAATT ATACCTGTGA  651 ATTAAAATAT GGAGGCTTTG TTGTGAGAAG AACTACTGAATTAACTGTTA  701 CAGCCCCTCT GACTGATAAG CCACCCAAGC TTTTGTATCC TATGGAAAGT 751 AAACTGACAA TTCAGGAGAC CCAGCTGGGT GACTCTGCTA ATCTAACCTG  801CAGAGCTTTC TTTGGGTACA GCGGAGATGT CAGTCCTTTA ATTTACTGGA  851 TGAAAGGAGAAAAATTTATT GAAGATCTGG ATGAAAATCG AGTTTGGGAA  901 AGTGACATTA GAATTCTTAAGGAGCATCTT GGGGAACAGG AAGTTTCCAT  951 CTCATTAATT GTGGACTCTG TGGAAGAAGGTGACTTGGGA AATTACTCCT 1001 GTTATGTTGA AAATGGAAAT GGACGTCGAC ACGCCAGCGTTCTCCTTCAT 1051 AAACGAGAGC TAATGTACAC AGTGGAACTT GCTGGAGGCC TTGGTGCTAT1101 ACTCTTGCTG CTTGTATGTT TGGTGACCAT CTACAAGTGT TACAAGATAG 1151AAATCATGCT CTTCTACAGG AATCATTTTG GAGCTGAAGA GCTCGATGGA 1201 GACAATAAAGATTATGATGC ATACTTATCA TACACCAAAG TGGATCCTGA 1251 CCAGTGGAAT CAAGAGACTGGGGAAGAAGA ACGTTTTGCC CTTGAAATCC 1301 TACCTGATAT GCTTGAAAAG CATTATGGATATAAGTTGTT TATACCAGAT 1351 AGAGATTTAA TCCCAACTGG AACATACATT GAAGATGTGGCAAGATGTGT 1401 AGATCAAAGC AAGCGGCTGA TTATTGTCAT GACCCCAAAT TACGTAGTTA1451 GAAGGGGCTG GAGCATCTTT GAGCTGGAAA CCAGACTTCG AAATATGCTT 1501GTGACTGGAG AAATTAAAGT GATTCTAATT GAATGCAGTG AACTGAGAGG 1551 AATTATGAACTACCAGGAGG TGGAGGCCCT GAAGCACACC ATCAAGCTCC 1601 TGACGGTCAT TAAATGGCATGGACCAAAAT GCAACAAGTT GAACTCCAAG 1651 TTCTGGAAAC GTTTACAGTA TGAAATGCCTTTTAAGAGGA TAGAACCCAT 1701 TACACATGAG CAGGCTTTAG ATGTCAGTGA GCAAGGGCCTTTTGGGGAGC 1751 TGCAGACTGT CTCGGCCATT TCCATGGCCG CGGCCACCTC CACAGCTCTA1801 GCCACTGCCC ATCCAGATCT CCGTTCTACC TTTCACAACA CGTACCATTC 1851ACAAATGCGT CAGAAACACT ACTACCGAAG CTATGAGTAC GACGTACCTC 1901 CTACCGGCACCCTGCCTCTT ACCTCCATAG GCAATCAGCA TACCTACTGT 1951 AACATCCCTA TGACACTCATCAACGGGCAG CGGCCACAGA CAAAATCGAG 2001 CAGGGAGCAG AATCCAGATG AGGCCCACACAAACAGTGCC ATCCTGCCGC 2051 TGTTGCCAAG GGAGACCAGT ATATCCAGTG TGATATGGTG AName: TDZ.1    1 ATGTCCTTTG TGGGGGAGAA CTCAGGAGTG AAAATGGGCT CTGAGGACTG(SEQ ID NO:5)   51 GGAAAAAGAT GAACCCCAGT GCTGCTTAGA AGACCCGGCTGTAAGCCCCC  101 TGGAACCAGG CCCAAGCCTC CCCACCATGA ATTTTGTTCA CACAAGTCCA 151 AAGGTGAAGA ACTTAAACCC GAAGAAATTC AGCATTCATG ACCAGGATCA  201CAAAGTACTG GTCCTGGACT CTGGGAATCT CATAGCAGTT CCAGATAAAA  251 ACTACATACGCCCAGAGATC TTCTTTGCAT TAGCCTCATC CTTGAGCTCA  301 GCCTCTGCGG AGAAAGGAAGTCCGATTCTC CTGGGGGTCT CTAAAGGGGA  351 GTTTTGTCTC TACTGTGACA AGGATAAAGGACAAAGTCAT CCATCCCTTC  401 AGCTGAAGAA GGAGAAACTG ATGAAGCTGG CTGCCCAAAAGGAATCAGCA  451 CGCCGGCCCT TCATCTTTTA TAGGGCTCAG GTGGGCTCCT GGAACATGCT 501 GGAGTCGGCG GCTCACCCCG GATGGTTCAT CTGCACCTCC TGCAATTGTA  551ATGAGCCTGT TGGGGTGACA GATAAATTTG AGAACAGGAA ACACATTGAA  601 TTTTCATTTCAACCAGTTTG CAAAGCTGAA ATGAGCCCCA GTGAGGTCAG  651 CGATTAG Name: TDZ.2   1 ATGTCCTTTG TGGGGGAGAA CTCAGGAGTG AAAATGGGCT CTGAGGACTG (SEQ IDNO:6)   51 GGAAAAAGAT GAACCCCAGT GCTGCTTAGA AGGTCCAAAG GTGAAGAACT  101TAAACCCGAA GAAATTCAGC ATTCATGACC AGGATCACAA AGTACTGGTC  151 CTGGACTCTGGGAATCTCAT AGCAGTTCCA GATAAAAACT ACATACGCCC  201 AGAGATCTTC TTTGCATTAGCCTCATCCTT GAGCTCAGCC TCTGCGGAGA  251 AAGGAAGTCC GATTCTCCTG GGGGTCTCTAAAGGGGAGTT TTGTCTCTAC  301 TGTGACAAGG ATAAAGGACA AAGTCATCCA TCCCTTCAGCTGAAGAAGGA  351 GAAACTGATG AAGCTGGCTG CCCAAAAGGA ATCAGCACGC CGGCCCTTCA 401 TCTTTTATAG GGCTCAGGTG GGCTCCTGGA ACATGCTGGA GTCGGCGGCT  451CACCCCGGAT GGTTCATCTG CACCTCCTGC AATTGTAATG AGCCTGTTGG  501 GGTGACAGATAAATTTGAGA ACAGGAAACA CATTGAATTT TCATTTCAAC  551 CAGTTTGCAA AGCTGAAATGAGCCCCAGTG AGGTCAGCGA TTAG

[0033] The amino acid sequences of the polypeptides encoded by thenucleotide sequence of the invention include: Name: IL-1 zeta(polypeptide)   1 MSGCDRRETE TKGKNSFKKR LRGPKVKNLN PKKFSIHDQDHKVLVLIDSGN (SEQ ID NO:3)  51 LIAVPDKNYI RPEIFFALAS SLSSASAEKGSPILLGVSKG EFCLYCDKDK 101 GQSHPSLQLK KEKLMKLAAQ KESARRPFIF YRAQVGSWNMLESAAHPGWF 151 ICTSCNCNEP VGVTDKFENR KHIEFSFQPV CKAEMSPSEV SD* Name:Xrec2 (polypeptide)   1 MKAPIPHLIL LYATFTQSLK VVTKRGSADG CTDWSIDIKKYQVLVGEPVR (SEQ ID NO:4)  51 IKCALFYGYI RTNYSLAQSA GLSLMWYKSS GPGDFEEPIAFDGSRMSKEE 101 DSIWFRPTLL QDSGLYACVI RNSTYCMKVS ISLTVGENDT GLCYNSKMKY151 FEKAELSKSK EISCRDIEDF LLPTREPEIL WYKECRTKTW RPSIVFKRDT 201LLIREVREDD IGNYTCELKY GGFVVRRTTE LTVTAPLTDK PPKLLYPMES 251 KLTIQETQLGDSANLTCRAF FGYSGDVSPL IYWMKGEKFI EDLDENRVWE 301 SDIRILKEHL GEQEVSISLIVDSVEEGDLG NYSCYVENGN GRRHASVLLH 351 KRELMYTVEL AGGLGAILLL LVCLVTIYKCYKIEIMLFYR NHFGAEELDG 401 DNKDYDAYLS YTKVDPDQWN QETGEEERFA LEILPDMLEKHYGYKLFIPD 451 RDLIPTGTYI EDVARCVDQS KRLIIVMTPN YVVRRGWSIF ELETRLRNML501 VTGEIKVILI ECSELRGIMN YQEVEALKHT IKLLTVIKWH GPKCNKLNSK 551FWKRLQYEMP FKRIEPITHE QALDVSEQGP FGELQTVSAI SMAAATSTAL 601 ATAHPDLRSTFHNTYHSQMR QKHYYRSYEY DVPPTGTLPL TSIGNQHTYC 651 NIPMTLINGQ RPQTKSSREQNPDEAHTNSA ILPLLPRETS ISSVIW* TDZ.1 polypeptide   1 MSFVGENSGVKMGSEDWEKD EPQCCLEDPA VSPLEPGPSL PTMNFVHTSP (SEQ ID NO:8)  51 KVKNLNPKKFSIHDQDHKVL VLDSGNLIAV PDKNYIRPEI FFALASSLSS 101 ASAEKGSPIL LGVSKGEFCLYCDKDKGQSH PSLQLKKEKL MKLAAQKESA 151 RRPFIFYRAQ VGSWNMLESA AHPGWFICTSCNCNEPVGVT DKFENRKHIE 201 FSFQPVCKAE MSPSEVSD* Name: TDZ.2 polypeptide  1 MSFVGENSGV KMGSEDWEKD EPQCCLEGPK VKNLNPKKFS IHDQDHKVLV (SEQ ID NO:9) 51 LDSGNLIAVP DKNYIRPEIF FALASSLSSA SAEKGSPILL GVSKGEFCLY 101CDKDKGQSHP SLQLKKEKLM KLAAQKESAR RPFIFYRAQV GSWNMLESAA 151 HPGWFICTSCNCNEPVGVTD KFENRKHIEF SFQPVCKAEM SPSEVSD* Name: TDZ.3 polypeptide   1MSFVGENSGV KMGSEDWEKD EPQCCLEEIF FALASSLSSA SAEKGSPILL (SEQ ID NO:10) 51 GVSKGEFCLY CDKDKGQSHP SLQLKKEKLM KLAAQKESAR RPFIFYRAQV 101GSWNMLESAA HPGWFICTSC NCNEPVGVTD KFENRKHIEF SFQPVCKAEM 151 SPSEVSD*

[0034] The discovery of the IL-1 zeta, the IL-1 zeta splice variants(TDZ.1, TDZ.2, and TDZ.3) and the Xrec2 nucleic acids of the inventionenables the construction of expression vectors comprising nucleic acidsequences encoding the respective polypeptides and host cellstransfected or transformed with the expression vectors. The inventionalso enables the isolation and purification of biologically active IL-1zeta, the IL-1 zeta splice variants, and Xrec2 polypeptides andfragments thereof. In yet another embodiment, the nucleic acids oroligonucleotides thereof can be used as probes to identify nucleic acidencoding proteins having associated activities. Thus, IL-1 zeta and theIL-1 splice variants can be used to identify activities associated withIL-1 family ligands and Xrec2 can be used to identify activitiesassociated with IL-1 family receptors. In addition, the nucleic acids oroligonucleotides thereof of IL-1 zeta can be used to identify humanchromosomes 2 while those of Xrec2 can be used to identify humanchromosome X. Similarly, these nucleic acids or oligonucleotides thereofcan be used to map genes on human chromosomes 2 and X, respectively, andto identify genes associated with certain diseases, syndromes or otherhuman conditions associated with human chromosomes 2 and X. Thus, thenucleic acids or oligonucleotides thereof of IL-1 zeta, TDZ.1, TDZ.2,and TDZ.3 can be used to identify glaucoma, ectodermal dysplasia,insulin-dependent diabetes mellitus, wrinkly skin syndrome, T-cellleukemia/lymphoma, and tibial muscular dystrophy while the nucleic acidsor oligonucleotides thereof of Xrec2 can be used to identifyretinoschisis, lissencephaly, subcortical laminalheteropia, mentalretardation, cowchock syndrome, bazex syndrome, hypertrichosis,lymphoproliferative syndrome, immunodeficiency, Langer mesomelicdysplasia, and leukemia. Finally, single-stranded sense or antisenseoligonucleotides from these nucleic acids can be used to inhibitexpression of polynucleotides encoded by the IL-1 zeta and Xrec2 genes,respectively.

[0035] Further, the IL-1 zeta, TDZ.1, TDZ.2, TDZ.3 and Xrec2polypeptides and soluble fragments thereof can be used to activateand/or inhibit the activation of vascular endothelial cells andlymphocytes, induce and/or inhibit the induction of local tissuedestruction and fever (Janeway et al., 1996), inhibit and/or stimulatemacrophages and vascular endothelial cells to produce IL-6, induceand/or inhibit the induction of prostaglandins, nitric oxide synthetase,and metalloproteinases, and upregulate and/or inhibit the upregulationof molecules on the surface of vascular endothelial cells. In additionthese polypeptides and fragmented peptides can also be used to induceand/or inhibit the induction of inflammatory mediators such astranscription factors NF-KB and AP-1, MAP kinases JNK and p38, COX-2,iNOS, and all of the activities stimulated by these molecules.

[0036] In addition, these polypeptides and fragmented peptides can beused as molecular weight markers and as controls for peptidefragmentation, and the invention includes the kits comprising thesereagents. Finally, these polypeptides and fragments thereof can be usedto generate antibodies, and the invention includes the use of suchantibodies to purify IL-1 zeta and Xrec2 polypeptides.

[0037] Nucleic Acid Molecules

[0038] In a particular embodiment, the invention relates to certainisolated nucleotide sequences that are free from contaminatingendogenous material. A “nucleotide sequence” refers to a polynucleotidemolecule in the form of a separate fragment or as a component of alarger nucleic acid construct. The nucleic acid molecule has beenderived from DNA or RNA isolated at least once in substantially pureform and in a quantity or concentration enabling identification,manipulation, and recovery of its component nucleotide sequences bystandard biochemical methods (such as those outlined in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd sed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989)). Such sequences arepreferably provided and/or constructed in the form of an open readingframe uninterrupted by internal non-translated sequences, or introns,that are typically present in eukaryotic genes. Sequences ofnon-translated DNA can be present 5′ or 3′ from an open reading frame,where the same do not interfere with manipulation or expression of thecoding region.

[0039] Nucleic acid molecules of the invention include DNA in bothsingle-stranded and double-stranded form, as well as the RNA complementthereof. DNA includes, for example, cDNA, genomic DNA, chemicallysynthesized DNA, DNA amplified by PCR, and combinations thereof. GenomicDNA may be isolated by conventional techniques, e.g., using the cDNA ofSEQ ID NOs:1, 2, 5, 6, 7 or a suitable fragment thereof, as a probe.

[0040] The DNA molecules of the invention include full length genes aswell as polynucleotides and fragments thereof. The full length gene mayinclude the N-terminal signal peptide. Other embodiments include DNAencoding a soluble form, e.g., encoding the extracellular domain of theprotein, either with or without the signal peptide.

[0041] The nucleic acids of the invention are preferentially derivedfrom human sources, but the invention includes those derived fromnon-human species, as well.

[0042] Preferred Sequences

[0043] The particularly preferred nucleic acid molecules of theinvention are those shown in SEQ ID NOs:1, 5, 6, 7 for IL-1 zeta, TDZ.1,TDZ.2, and TDZ.3, respectively, and SEQ ID NO:2 for Xrec2. cDNA cloneshaving the nucleic acid sequence of SEQ ID NOs:1 and 2 were isolated asdescribed in Example 1. The sequences of the amino acids of IL-1 zetaand Xrec2 encoded by the DNAs of SEQ ID NOs:1 and 2 are shown in SEQ IDNOs:3 and 4, respectively. cDNA clones having the nucleic acid sequenceof SEQ ID NOs:5, 6, and 7 were isolated as described in Example 8. Thesequences of the amino acids of TDZ.1, TDZ.2, and TDZ.3 encoded by theDNAs of SEQ ID NOs:5, 6, and 7 are shown in SEQ ID NOs:8, 9, and 10,respectively.

[0044] SEQ ID NOs:1-4 identify the IL-1 zeta of SEQ ID NO:3 as a memberof the IL-1 family and the Xrec2 of SEQ ID NO:4 as a member of the IL-1receptor family. The homologies on which this is based is set forth atTable I below: TABLE I Protein Source Percent identity to IL-1 zeta IL-1alpha Human LOW IL-1 beta Human 22% IL-1 delta Human 34% IL-1 epsilonHuman 34% IL-18 Human LOW IL-1ra Human 29% Percent identity to Xrec2TIGIRR (IL-1R family member) Human 63% TIGIRR (IL-1R family member)Murine 61% SIGLRR Human 22% IL-1R-AcP Human 35% IL-1R-AcPL Human 26%IL-1R Human 29% RP1 Human 31% RP2 Human 28% ST2 Human 26%

[0045] Percent identity of IL-1 zeta and Xrec2 to human and murineproteins.

[0046] As described in Example 8, the IL-1 zeta splice variants werediscovered in a stretch of genomic DNA sequence (X22304.gbn). Thisgenomic sequence also contains the different IL-1 zeta exons and anothersplice variant known as Tango-77 (WO 99/06426). Comparing the cDNAsequences of the cloned IL-1 zeta, TDZ.1, TDZ.2, TDZ.3 and Tango-77 withthe genomic sequence provides insight into the generation of thesplicing events. FIG. 1 shows the genomic structure of the IL-1 zetalocus and the cDNA generated by alternative splicing. The numbered boxesindicate individual exons 1-6 and the approximate size of theintervening introns is indicated at the top. The asterisk (*) indicatesthe presence of a stop codon, at the end of the coding sequence (exon 6)or as an in-frame stop codon (exon 3). “M” indicates potentialinitiating methionine originating either from exon 1 or exon 3. Tango-77is the cDNA structure disclosed in WO 99/06426. A significant feature ofIL-1 zeta and its splice variants is the presence or the absence of exon4. Exon 4 is present in IL-1 zeta, TDZ.1 and TDZ.2. It is not present inTango-77. The amino acid sequence encoded by exon 4 aligns well with theamino acid sequences of other IL-1 family members in the first few betastrands of the mature peptides. By contrast, the amino acid sequenceencoded by Tango-77 cDNA and by TDZ.3 cDNA aligns well with other IL-1family members in the regions encoded by exons 5 and 6. Exons 5 and 6align well with amino acid sequences of other IL-1 family members in theC-terminal 2/3 of the mature peptide, but does not align well in theN-terminal 1/3. Thus, the “mature peptide” encoded by IL1zeta, TDZ.1 andTDZ.2 DNAs is likely to represent a functional IL-1 like molecule. Thiscontrasts with the polypeptide encoded by Tango-77 or TDZ.3 DNAS whichare less likely to represent a functional IL-1 like molecule.

[0047] It is probable that all of the splice isoforms, except TDZ.3,encode proforms of an IL-1 like cytokine, since in the N-terminaldirection the DNAs extend well beyond the N-terminus of mature IL-1s.This observation predicts that IL-1zeta, TDZ.1 and TDZ.2 encode the samemature peptide. In connection with this observation it is thepro-domains (as well as 5′ UTRs) that differs between IL-1 zeta, TDZ.1and TDZ.2.

[0048] Table II, which details the tissue distribution of IL-1 zeta,TDZ.1, TDZ.2, TDZ.3 and Tango-77, shows that the expression of Tango-77is more widespread than that of IL-1 zeta. Table II also shows that theTDZ.1 expression is comparable, and almost entirely overlapping, withthat of Tango-77. The tissue distribution data combined with thealignment information of FIG. 1 shows that TDZ.1 is the only member ofthe splice variants that aligns well with other IL-1 family members, andis widespread in its expression. These observations suggest that TDZ.1may be the most significant of the splice variants in terms of group interms of relevance to biological responses.

[0049] Additional Sequences

[0050] Due to the known degeneracy of the genetic code, wherein morethan one codon can encode the same amino acid, a DNA sequence can varyfrom that shown in SEQ ID NOs:1, 2, 5, 6, and 7 and still encode apolypeptide having the amino acid sequence of SEQ ID NOs:3, 4, 8, 9, and10, respectively. Such variant DNA sequences can result from silentmutations (e.g., occurring during PCR amplification), or can be theproduct of deliberate mutagenesis of a native sequence.

[0051] The invention thus provides isolated DNA sequences encodingpolypeptides of the invention, selected from: (a) DNA comprising thenucleotide sequences of SEQ ID NOs:1, 2; 5, 6, and 7 (b) DNA encodingthe polypeptides of SEQ ID NOs:3, 4; 8, 9, and 10 (c) DNA capable ofhybridization to a DNA of (a) or (b) under conditions of moderatestringency and which encodes polypeptides of the invention; (d) DNAcapable of hybridization to a DNA of (a) or (b) under conditions of highstringency and which encodes polypeptides of the invention, and (e) DNAwhich is degenerate, as a result of the genetic code, to a DNA definedin (a), (b), (c), or (d) and which encode polypeptides of the invention.Of course, polypeptides encoded by such DNA sequences are encompassed bythe invention.

[0052] As used herein, conditions of moderate stringency can be readilydetermined by those having ordinary skill in the art based on, forexample, the length of the DNA. The basic conditions are set forth bySambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1,pp. 1.101-104, Cold Spring Harbor Laboratory Press, (1989), and includeuse of a prewashing solution for the nitrocellulose filters 5×SSC, 0.5%SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of about 50%formamide, 6×SSC at about 42° C. (or other similar hybridizationsolution, such as Stark's solution, in about 50% formamide at about 42°C.), and washing conditions of about 60° C., 0.5×SSC, 0.1% SDS.Conditions of high stringency can also be readily determined by theskilled artisan based on, for example, the length of the DNA. Generally,such conditions are defined as hybridization conditions as above, andwith washing at approximately 68° C., 0.2×SSC, 0.1% SDS. The skilledartisan will recognize that the temperature and wash solution saltconcentration can be adjusted as necessary according to factors such asthe length of the probe.

[0053] Also included as an embodiment of the invention is DNA encodingpolypeptide fragments and polypeptides comprising inactivatedN-glycosylation site(s), inactivated protease processing site(s), orconservative amino acid substitution(s), as described below.

[0054] In another embodiment, the nucleic acid molecules of theinvention also comprise nucleotide sequences that are at least 80%identical to a native sequence. Also contemplated are embodiments inwhich a nucleic acid molecule comprises a sequence that is at least 90%identical, at least 95% identical, at least 98% identical, at least 99%identical, or at least 99.9% identical to a native sequence.

[0055] The percent identity may be determined by visual inspection andmathematical calculation. Alternatively, the percent identity of twonucleic acid sequences can be determined by comparing sequenceinformation using the GAP computer program, version 6.0 described byDevereux et al. (Nucl. Acids Res. 12:387, 1984) and available from theUniversity of Wisconsin Genetics Computer Group (UWGCG). The preferreddefault parameters for the GAP program include: (1) a unary comparisonmatrix (containing a value of 1 for identities and 0 for non-identities)for nucleotides, and the weighted comparison matrix of Gribskov andBurgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz andDayhoff, eds., Atlas of Protein Sequence and Structure, NationalBiomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0for each gap and an additional 0.10 penalty for each symbol in each gap;and (3) no penalty for end gaps. Other programs used by one skilled inthe art of sequence comparison may also be used.

[0056] The invention provides isolated nucleic acids useful in theproduction of polypeptides. Such polypeptides may be prepared by any ofa number of conventional techniques. A DNA sequence encoding apolypeptide of the invention, or desired fragment thereof may besubcloned into an expression vector for production of the polypeptide orfragment. The DNA sequence advantageously is fused to a sequenceencoding a suitable leader or signal peptide. Alternatively, the desiredfragment may be chemically synthesized using known techniques. DNAfragments also may be produced by restriction endonuclease digestion ofa full length cloned DNA sequence, and isolated by electrophoresis onagarose gels. If necessary, oligonucleotides that reconstruct the 5′ or3′ terminus to a desired point may be ligated to a DNA fragmentgenerated by restriction enzyme digestion. Such oligonucleotides mayadditionally contain a restriction endonuclease cleavage site upstreamof the desired coding sequence, and position an initiation codon (ATG)at the N-terminus of the coding sequence.

[0057] The well-known polymerase chain reaction (PCR) procedure also maybe employed to isolate and amplify a DNA sequence encoding a desiredprotein fragment. Oligonucleotides that define the desired termini ofthe DNA fragment are employed as 5′ and 3′ primers. The oligonucleotidesmay additionally contain recognition sites for restrictionendonucleases, to facilitate insertion of the amplified DNA fragmentinto an expression vector. PCR techniques are described in Saiki et al.,Science 239:487 (1988); Recombinant DNA Methodology, Wu et al., eds.,Academic Press, Inc., San Diego (1989), pp. 189-196; and PCR Protocols:A Guide to Methods and Applications, Innis et al., eds., Academic Press,Inc. (1990).

[0058] Polypeptides and Fragments Thereof

[0059] The invention encompasses polypeptides and fragments thereof invarious forms, including those that are naturally occurring or producedthrough various techniques such as procedures involving recombinant DNAtechnology. Such forms include, but are not limited to, derivatives,variants, and oligomers, as well as fusion proteins or fragmentsthereof.

[0060] The polypeptides of the invention include full length proteinsencoded by the nucleic acid sequences set forth above. Particularlypreferred polypeptides of IL-1 zeta, TDZ.1, TDZ.2 TDZ.3 and Xrec2comprise the amino acid sequence of SEQ ID NOs:3, 4, 8, 9, and 10respectively. For TDZ.1 and TDZ.2 the N-terminus does not encode aclassical signal peptide but the extra length relative to the matureform other family members is suggestive that it may act as a prodomain.A predicted cleavage site is the point where the conserved structuralportion of the protein begins. Structural modeling data supports thisassumption. For IL-1 zeta and the TDZ.1 and TDZ.2 variants site issomewhere immediately upstream of the last three exons. Thus, thepolypeptide of IL-1 zeta, as set forth in SEQ ID NO:3, includes aputative pro-domain that extends from amino acids 1 to x, where x is aninteger from 20 to 50. Similarly, TDZ.1 of SEQ ID NO:8 includes aputative prodomain that extends from amino acids 1 to x′ where x′ is aninteger from 40-50 and most preferably x′ is about 48. TDZ.2 of SEQ IDNO:9 includes a putative prodomain that extends from amino acids 1 tox″, where x″ is an integer from 25-30 and most preferable x″ is 27.

[0061] Unlike IL-1 zeta and its splice variants, the polypeptide ofXrec2, as set forth in SEQ ID NO:4, includes an N-terminal hydrophobicregion that functions as a signal peptide, followed by an extracellulardomain comprising amino acids 19 to 359, a transmembrane regioncomprising amino acids 360 through 378, and a C-terminal cytoplasmicdomain comprising amino acids 379 to 696. Computer analysis predictsthat the signal peptide corresponds to residues 1 to 19 of SEQ ID NO:4(although the next most likely computer-predicted signal peptidecleavage sites (in descending order) occur after amino acids 20 and 16of SEQ ID NO:4.)). Cleavage of the signal peptide thus would yield amature protein comprising amino acids 19 through 696 of SEQ ID NO:4.

[0062] The skilled artisan will recognize that the above-describedboundaries of such regions of the polypeptide are approximate. Toillustrate, the boundaries of the transmembrane region (which may bepredicted by using computer programs available for that purpose) maydiffer from those described above.

[0063] The polypeptides of the invention may be membrane bound or theymay be secreted and, thus, soluble. Soluble polypeptides are capable ofbeing secreted from the cells in which they are expressed. In general,soluble polypeptides may be identified (and distinguished fromnon-soluble membrane-bound counterparts) by separating intact cellswhich express the desired polypeptide from the culture medium, e.g., bycentrifugation, and assaying the medium (supernatant) for the presenceof the desired polypeptide. The presence of polypeptide in the mediumindicates that the polypeptide was secreted from the cells and thus is asoluble form of the protein.

[0064] In one embodiment, the soluble polypeptides and fragments thereofcomprise all or part of the extracellular domain, but lack thetransmembrane region that would cause retention of the polypeptide on acell membrane. A soluble polypeptide may include the cytoplasmic domain,or a portion thereof, as long as the polypeptide is secreted from thecell in which it is produced.

[0065] In general, the use of soluble forms is advantageous for certainapplications. Purification of the polypeptides from recombinant hostcells is facilitated, since the soluble polypeptides are secreted fromthe cells. Further, soluble polypeptides are generally more suitable forintravenous administration.

[0066] The invention also provides polypeptides and fragments of theextracellular domain that retain a desired biological activity.Particular embodiments are directed to polypeptide fragments of SEQ IDNOs:3, 4, 8, 9, and 10 that retain the ability to bind the nativecognates, substrates, or counter-structure (“binding partner”). Such afragment may be a soluble polypeptide, as described above. In anotherembodiment, the polypeptides and fragments advantageously includeregions that are conserved in the IL-1 ligand and IL-1 receptor familyas described above.

[0067] Also provided herein are polypeptide fragments comprising atleast 20, or at least 30, contiguous amino acids of the sequences of SEQID NOs:3 4, 8, 9, and 10. In one aspect, fragments derived from thecytoplasmic domain of Xrec2 of SEQ ID NO:4 find use in studies of signaltransduction, and in regulating cellular processes associated withtransduction of biological signals. Polypeptide fragments also may beemployed as immunogens, in generating antibodies.

[0068] Variants

[0069] Naturally occurring variants as well as derived variants of thepolypeptides and fragments are provided herein.

[0070] Variants may exhibit amino acid sequences that are at least 80%identical. Also contemplated are embodiments in which a polypeptide orfragment comprises an amino acid sequence that is at least 90%identical, at least 95% identical, at least 98% identical, at least 99%identical, or at least 99.9% identical to the preferred polypeptide orfragment thereof. Percent identity may be determined by visualinspection and mathematical calculation. Alternatively, the percentidentity of two protein sequences can be determined by comparingsequence information using the GAP computer program, based on thealgorithm of Needleman and Wunsch (J. Mol. Bio. 48:443, 1970) andavailable from the University of Wisconsin Genetics Computer Group(UWGCG). The preferred default parameters for the GAP program include:(1) a scoring matrix, blosum62, as described by Henikoff and Henikoff(Proc. Natl. Acad. Sci. USA 89:10915, 1992); (2) a gap weight of 12; (3)a gap length weight of 4; and (4) no penalty for end gaps. Otherprograms used by one skilled in the art of sequence comparison may alsobe used.

[0071] The variants of the invention include, for example, those thatresult from alternate mRNA splicing events or from proteolytic cleavage.Alternate splicing of mRNA may, for example, yield a truncated butbiologically active protein, such as a naturally occurring soluble formof the protein. Variations attributable to proteolysis include, forexample, differences in the N- or C-termini upon expression in differenttypes of host cells, due to proteolytic removal of one or more terminalamino acids from the protein (generally from 1-5 terminal amino acids).Proteins in which differences in amino acid sequence are attributable togenetic polymorphism (allelic variation among individuals producing theprotein) are also contemplated herein.

[0072] Additional variants within the scope of the invention includepolypeptides that may be modified to create derivatives thereof byforming covalent or aggregative conjugates with other chemical moieties,such as glycosyl groups, lipids, phosphate, acetyl groups and the like.Covalent derivatives may be prepared by linking the chemical moieties tofunctional groups on amino acid side chains or at the N-terminus orC-terminus of a polypeptide. Conjugates comprising diagnostic(detectable) or therapeutic agents attached thereto are contemplatedherein, as discussed in more detail below.

[0073] Other derivatives include covalent or aggregative conjugates ofthe polypeptides with other proteins or polypeptides, such as bysynthesis in recombinant culture as N-terminal or C-terminal fusions.Examples of fusion proteins are discussed below in connection witholigomers. Further, fusion proteins can comprise peptides added tofacilitate purification and identification. Such peptides include, forexample, poly-His or the antigenic identification peptides described inU.S. Pat. No. 5,011,912 and in Hopp et al., BioTechnology 6:1204, 1988.One such peptide is the FLAG⁷ peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys,which is highly antigenic and provides an epitope reversibly bound by aspecific monoclonal antibody, enabling rapid assay and facilepurification of expressed recombinant protein. A murine hybridomadesignated 4E11 produces a monoclonal antibody that binds the FLAG⁷peptide in the presence of certain divalent metal cations, as describedin U.S. Pat. No. 5,011,912, hereby incorporated by reference. The 4E11hybridoma cell line has been deposited with the American Type CultureCollection under accession no. HB 9259. Monoclonal antibodies that bindthe FLAG⁷ peptide are available from Eastman Kodak Co., ScientificImaging Systems Division, New Haven, Conn.

[0074] Among the variant polypeptides provided herein are variants ofnative polypeptides that retain the native biological activity or thesubstantial equivalent thereof. One example is a variant that binds withessentially the same binding affinity as does the native form. Bindingaffinity can be measured by conventional procedures, e.g., as describedin U.S. Pat. No. 5,512,457 and as set forth below.

[0075] Variants include polypeptides that are substantially homologousto the native form, but which have an amino acid sequence different fromthat of the native form because of one or more deletions, insertions orsubstitutions. Particular embodiments include, but are not limited to,polypeptides that comprise from one to ten deletions, insertions orsubstitutions of amino acid residues, when compared to a nativesequence.

[0076] A given amino acid may be replaced, for example, by a residuehaving similar physiochemical characteristics. Examples of suchconservative substitutions include substitution of one aliphatic residuefor another, such as Ile, Val, Leu, or Ala for one another;substitutions of one polar residue for another, such as between Lys andArg, Glu and Asp, or Gln and Asn; or substitutions of one aromaticresidue for another, such as Phe, Trp, or Tyr for one another. Otherconservative substitutions, e.g., involving substitutions of entireregions having similar hydrophobicity characteristics, are well known.

[0077] Similarly, the DNAs of the invention include variants that differfrom a native DNA sequence because of one or more deletions, insertionsor substitutions, but that encode a biologically active polypeptide.

[0078] The invention further includes polypeptides of the invention withor without associated native-pattern glycosylation. Polypeptidesexpressed in yeast or mammalian expression systems (e.g., COS-1 or COS-7cells) can be similar to or significantly different from a nativepolypeptide in molecular weight and glycosylation pattern, dependingupon the choice of expression system. Expression of polypeptides of theinvention in bacterial expression systems, such as E. coli, providesnon-glycosylated molecules. Further, a given preparation may includemultiple differentially glycosylated species of the protein. Glycosylgroups can be removed through conventional methods, in particular thoseutilizing glycopeptidase. In general, glycosylated polypeptides of theinvention can be incubated with a molar excess of glycopeptidase(Boehringer Mannheim).

[0079] Correspondingly, similar DNA constructs that encode variousadditions or substitutions of amino acid residues or sequences, ordeletions of terminal or internal residues or sequences are encompassedby the invention. For example, N-glycosylation sites in the polypeptideextracellular domain can be modified to preclude glycosylation, allowingexpression of a reduced carbohydrate analog in mammalian and yeastexpression systems. N-glycosylation sites in eukaryotic polypeptides arecharacterized by an amino acid triplet Asn-X-Y, wherein X is any aminoacid except Pro and Y is Ser or Thr. Appropriate substitutions,additions, or deletions to the nucleotide sequence encoding thesetriplets will result in prevention of attachment of carbohydrateresidues at the Asn side chain. Alteration of a single nucleotide,chosen so that Asn is replaced by a different amino acid, for example,is sufficient to inactivate an N-glycosylation site. Alternatively, theSer or Thr can by replaced with another amino acid, such as Ala. Knownprocedures for inactivating N-glycosylation sites in proteins includethose described in U.S. Pat. No. 5,071,972 and EP 276,846, herebyincorporated by reference.

[0080] In another example of variants, sequences encoding Cys residuesthat are not essential for biological activity can be altered to causethe Cys residues to be deleted or replaced with other amino acids,preventing formation of incorrect intramolecular disulfide bridges uponfolding or renaturation.

[0081] Other variants are prepared by modification of adjacent dibasicamino acid residues, to enhance expression in yeast systems in whichKEX2 protease activity is present. EP 212,914 discloses the use ofsite-specific mutagenesis to inactivate KEX2 protease processing sitesin a protein. KEX2 protease processing sites are inactivated bydeleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, andLys-Arg pairs to eliminate the occurrence of these adjacent basicresidues. Lys-Lys pairings are considerably less susceptible to KEX2cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents aconservative and preferred approach to inactivating KEX2 sites.

[0082] Oligomers

[0083] Encompassed by the invention are oligomers or fusion proteinsthat contain IL-1 zeta, TDZ.1, TDZ.2, TDZ.3 or Xrec2 polypeptides. Whenthe polypeptide of the invention is a type I membrane protein, such asXrec2, the fusion partner is linked to the C terminus of the type Imembrane protein. Such oligomers may be in the form of covalently-linkedor non-covalently-linked multimers, including dimers, trimers, or higheroligomers. As noted above, preferred polypeptides are soluble and thusthese oligomers may comprise soluble polypeptides. In one aspect of theinvention, the oligomers maintain the binding ability of the polypeptidecomponents and provide therefor, bivalent, trivalent, etc., bindingsites.

[0084] One embodiment of the invention is directed to oligomerscomprising multiple polypeptides joined via covalent or non-covalentinteractions between peptide moieties fused to the polypeptides. Suchpeptides may be peptide linkers (spacers), or peptides that have theproperty of promoting oligomerization. Leucine zippers and certainpolypeptides derived from antibodies are among the peptides that canpromote oligomerization of the polypeptides attached thereto, asdescribed in more detail below.

[0085] Immunoglobulin-Based Oligomers

[0086] As one alternative, an oligomer is prepared using polypeptidesderived from immunoglobulins. Preparation of fusion proteins comprisingcertain heterologous polypeptides fused to various portions ofantibody-derived polypeptides (including the Fc domain) has beendescribed, e.g., by Ashkenazi et al. (PNAS USA 88:10535, 1991); Byrn etal. (Nature 344:677, 1990); and Hollenbaugh and Aruffo (“Construction ofImmunoglobulin Fusion Proteins”, in Current Protocols in Immunology,Suppl. 4, pages 10.19.1-10.19.11, 1992).

[0087] One embodiment of the present invention is directed to a dimercomprising two fusion proteins created by fusing a polypeptide of theinvention to an Fc polypeptide derived from an antibody. A gene fusionencoding the polypeptide/Fc fusion protein is inserted into anappropriate expression vector. Polypeptide/Fc fusion proteins areexpressed in host cells transformed with the recombinant expressionvector, and allowed to assemble much like antibody molecules, whereuponinterchain disulfide bonds form between the Fc moieties to yielddivalent molecules.

[0088] The term “Fc polypeptide” as used herein includes native andmutein forms of polypeptides made up of the Fc region of an antibodycomprising any or all of the CH domains of the Fc region. Truncatedforms of such polypeptides containing the hinge region that promotesdimerization are also included. Preferred polypeptides comprise an Fcpolypeptide derived from a human IgG1 antibody.

[0089] One suitable Fc polypeptide, described in PCT application WO93/10151, hereby incorporated by reference, is a single chainpolypeptide extending from the N-terminal hinge region to the nativeC-terminus of the Fc region of a human IgG1 antibody. Another useful Fcpolypeptide is the Fc mutein described in U.S. Pat. No. 5,457,035 and inBaum et al., (EMBO J. 13:3992-4001, 1994) incorporated herein byreference. The amino acid sequence of this mutein is identical to thatof the native Fc sequence presented in WO 93/10151, except that aminoacid 19 has been changed from Leu to Ala, amino acid 20 has been changedfrom Leu to Glu, and amino acid 22 has been changed from Gly to Ala. Themutein exhibits reduced affinity for Fc receptors.

[0090] The above-described fusion proteins comprising Fc moieties (andoligomers formed therefrom) offer the advantage of facile purificationby affinity chromatography over Protein A or Protein G columns.

[0091] In other embodiments, the polypeptides of the invention may besubstituted for the variable portion of an antibody heavy or lightchain. If fusion proteins are made with both heavy and light chains ofan antibody, it is possible to form an oligomer with as many as fourpolypeptide extracellular regions.

[0092] Peptide-linker Based Oligomers

[0093] Alternatively, the oligomer is a fusion protein comprisingmultiple polypeptides, with or without peptide linkers (spacerpeptides). Among the suitable peptide linkers are those described inU.S. Pat. Nos. 4,751,180 and 4,935,233, which are hereby incorporated byreference. A DNA sequence encoding a desired peptide linker may beinserted between, and in the same reading frame as, the DNA sequences ofthe invention, using any suitable conventional technique. For example, achemically synthesized oligonucleotide encoding the linker may beligated between the sequences. In particular embodiments, a fusionprotein comprises from two to four soluble polypeptides of theinvention, separated by peptide linkers.

[0094] Leucine-Zippers

[0095] Another method for preparing the oligomers of the inventioninvolves use of a leucine zipper. Leucine zipper domains are peptidesthat promote oligomerization of the proteins in which they are found.Leucine zippers were originally identified in several DNA-bindingproteins (Landschulz et al., Science 240:1759, 1988), and have sincebeen found in a variety of different proteins. Among the known leucinezippers are naturally occurring peptides and derivatives thereof thatdimerize or trimerize.

[0096] The zipper domain (also referred to herein as an oligomerizing,or oligomer-forming, domain) comprises a repetitive heptad repeat, oftenwith four or five leucine residues interspersed with other amino acids.Examples of zipper domains are those found in the yeast transcriptionfactor GCN4 and a heat-stable DNA-binding protein found in rat liver(C/EBP; Landschulz et al., Science 243:1681, 1989). Two nucleartransforming proteins, fos and jun, also exhibit zipper domains, as doesthe gene product of the murine proto-oncogene, c-myc (Landschulz et al.,Science 240:1759, 1988). The products of the nuclear oncogenes fos andjun comprise zipper domains that preferentially form heterodimers(O'Shea et al., Science 245:646, 1989, Turner and Tjian, Science243:1689, 1989). The zipper domain is necessary for biological activity(DNA binding) in these proteins.

[0097] The fusogenic proteins of several different viruses, includingparamyxovirus, coronavirus, measles virus and many retroviruses, alsopossess zipper domains (Buckland and Wild, Nature 338:547,1989; Britton,Nature 353:394, 1991; Delwart and Mosialos, AIDS Research and HumanRetroviruses 6:703, 1990). The zipper domains in these fusogenic viralproteins are near the transmembrane region of the proteins; it has beensuggested that the zipper domains could contribute to the oligomericstructure of the fusogenic proteins. Oligomerization of fusogenic viralproteins is involved in fusion pore formation (Spruce et al, Proc. Natl.Acad. Sci. U.S.A. 88:3523, 1991). Zipper domains have also been recentlyreported to play a role in oligomerization of heat-shock transcriptionfactors (Rabindran et al., Science 259:230, 1993).

[0098] Zipper domains fold as short, parallel coiled coils. (O'Shea etal., Science 254:539; 1991) The general architecture of the parallelcoiled coil has been well characterized, with a “knobs-into-holes”packing as proposed by Crick in 1953 (Acta Crystallogr. 6:689). Thedimer formed by a zipper domain is stabilized by the heptad repeat,designated (abcdefg)_(n) according to the notation of McLachlan andStewart (J. Mol. Biol. 98:293; 1975), in which residues a and d aregenerally hydrophobic residues, with d being a leucine, which line up onthe same face of a helix. Oppositely-charged residues commonly occur atpositions g and e. Thus, in a parallel coiled coil formed from twohelical zipper domains, the “knobs” formed by the hydrophobic sidechains of the first helix are packed into the “holes” formed between theside chains of the second helix.

[0099] The residues at position d (often leucine) contribute largehydrophobic stabilization energies, and are important for oligomerformation (Krystek: et al., Int. J. Peptide Res. 38:229, 1991). Lovejoyet al. (Science 259:1288, 1993) recently reported the synthesis of atriple-stranded α-helical bundle in which the helices run up-up-down.Their studies confirmed that hydrophobic stabilization energy providesthe main driving force for the formation of coiled coils from helicalmonomers. These studies also indicate that electrostatic interactionscontribute to the stoichiometry and geometry of coiled coils. Furtherdiscussion of the structure of leucine zippers is found in Harbury etal. (Science 262:1401, Nov. 26, 1993)

[0100] Examples of leucine zipper domains suitable for producing solubleoligomeric proteins are described in PCT application WO 94/10308, andthe leucine zipper derived from lung surfactant protein D (SPD)described in Hoppe et al. (FEBS Letters 344:191, 1994), herebyincorporated by reference. The use of a modified leucine zipper thatallows for stable trimerization of a heterologous protein fused theretois described in Fanslow et al. (Semin. Immunol. 6:267-278, 1994).Recombinant fusion proteins comprising a soluble polypeptide fused to aleucine zipper peptide are expressed in suitable host cells, and thesoluble oligomer that forms is recovered from the culture supernatant.

[0101] Certain leucine zipper moieties preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D(SPD), as described in Hoppe et al. (FEBS Letters 344:191, 1994) and inU.S. Pat. No. 5,716,805, hereby incorporated by reference in theirentirety. This lung SPD-derived leucine zipper peptide comprises theamino acid sequence Pro Asp Val Ala Ser Leu Arg Gln Gln Val Glu Ala LeuGln Gly Gln Val Gln His Leu Gln Ala Ala Phe Ser Gln Tyr.

[0102] Another example of a leucine zipper that promotes trimerizationis a peptide comprising the amino acid sequence Arg Met Lys Gln Ile GluAsp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile AlaArg Ile Lys Lys Leu Ile Gly Glu Arg, as described in U.S. Pat. No.5,716,805. In one alternative embodiment, an N-terminal Asp residue isadded; in another, the peptide lacks the N-terminal Arg residue.

[0103] Fragments of the foregoing zipper peptides that retain theproperty of promoting oligomerization may be employed as well. Examplesof such fragments include, but are not limited to, peptides lacking oneor two of the N-terminal or C-terminal residues presented in theforegoing amino acid sequences. Leucine zippers may be derived fromnaturally occurring leucine zipper peptides, e.g., via conservativesubstitution(s) in the native amino acid sequence, wherein the peptide'sability to promote oligomerization is retained.

[0104] Other peptides derived from naturally occurring trimeric proteinsmay be employed in preparing trimeric oligomers. Alternatively,synthetic peptides that promote oligomerization may be employed. Inparticular embodiments, leucine residues in a leucine zipper moiety arereplaced by isoleucine residues. Such peptides comprising isoleucine maybe referred to as isoleucine zippers, but are encompassed by the term“leucine zippers” as employed herein.

[0105] Production of Polypeptides and Fragments Thereof

[0106] Expression, isolation and purification of the polypeptides andfragments of the invention may be accomplished by any suitabletechnique, including but not limited to the following:

[0107] Expression Systems

[0108] The present invention also provides recombinant cloning andexpression vectors containing DNA, as well as host cell containing therecombinant vectors. Expression vectors comprising DNA may be used toprepare the polypeptides or fragments of the invention encoded by theDNA. A method for producing polypeptides comprises culturing host cellstransformed with a recombinant expression vector encoding thepolypeptide, under conditions that promote expression of thepolypeptide, then recovering the expressed polypeptides from theculture. The skilled artisan will recognize that the procedure forpurifying the expressed polypeptides will vary according to such factorsas the type of host cells employed, and whether the polypeptide ismembrane-bound or a soluble form that is secreted from the host cell.

[0109] Any suitable expression system may be employed. The vectorsinclude a DNA encoding a polypeptide or fragment of the invention,operably linked to suitable transcriptional or translational regulatorynucleotide sequences, such as those derived from a mammalian, microbial,viral, or insect gene. Examples of regulatory sequences includetranscriptional promoters, operators, or enhancers, an mRNA ribosomalbinding site, and appropriate sequences which control transcription andtranslation initiation and termination. Nucleotide sequences areoperably linked when the regulatory sequence functionally relates to theDNA sequence. Thus, a promoter nucleotide sequence is operably linked toa DNA sequence if the promoter nucleotide sequence controls thetranscription of the DNA sequence. An origin of replication that confersthe ability to replicate in the desired host cells, and a selection geneby which transformants are identified, are generally incorporated intothe expression vector.

[0110] In addition, a sequence encoding an appropriate signal peptide(native or heterologous) can be incorporated into expression vectors. ADNA sequence for a signal peptide (secretory leader) may be fused inframe to the nucleic acid sequence of the invention so that the DNA isinitially transcribed, and the mRNA translated, into a fusion proteincomprising the signal peptide. A signal peptide that is functional inthe intended host cells promotes extracellular secretion of thepolypeptide. The signal peptide is cleaved from the polypeptide uponsecretion of polypeptide from the cell.

[0111] The skilled artisan will also recognize that the position(s) atwhich the signal peptide is cleaved may differ from that predicted bycomputer program, and may vary according to such factors as the type ofhost cells employed in expressing a recombinant polypeptide. A proteinpreparation may include a mixture of protein molecules having differentN-terminal amino acids, resulting from cleavage of the signal peptide atmore than one site. Particular embodiments of mature proteins providedherein include, but are not limited to, proteins having the residue atposition 6, 23, 25, 26, 39, 41, or 48 of SEQ ID NO:3 and at position 1or 19 of SEQ ID NO:4 as the N-terminal amino acid.

[0112] Suitable host cells for expression of polypeptides includeprokaryotes, yeast or higher eukaryotic cells. Mammalian or insect cellsare generally preferred for use as host cells. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described, for example, in Pouwels et al. CloningVectors: A Laboratory Manual, Elsevier, N.Y., (1985). Cell-freetranslation systems could also be employed to produce polypeptides usingRNAs derived from DNA constructs disclosed herein.

[0113] Prokaryotic Systems

[0114] Prokaryotes include gram-negative or gram-positive organisms.Suitable prokaryotic host cells for transformation include, for example,E. coli, Bacillus subtilis, Salmonella typhimurium, and various otherspecies within the genera Pseudomonas, Streptomyces, and Staphylococcus.In a prokaryotic host cell, such as E. coli, a polypeptide may includean N-terminal methionine residue to facilitate expression of therecombinant polypeptide in the prokaryotic host cell. The N-terminal Metmay be cleaved from the expressed recombinant polypeptide.

[0115] Expression vectors for use in prokaryotic host cells generallycomprise one or more phenotypic selectable marker genes. A phenotypicselectable marker gene is, for example, a gene encoding a protein thatconfers antibiotic resistance or that supplies an autotrophicrequirement. Examples of useful expression vectors for prokaryotic hostcells include those derived from commercially available plasmids such asthe cloning vector pBR322 (ATCC 37017). pBR322 contains genes forampicillin and tetracycline resistance and thus provides simple meansfor identifying transformed cells. An appropriate promoter and a DNAsequence are inserted into the pBR322 vector. Other commerciallyavailable vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis.,USA).

[0116] Promoter sequences commonly used for recombinant prokaryotic hostcell expression vectors include β-lactamase (penicillinase), lactosepromoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al.,Nature 281:544, 1979), tryptophan (trp) promoter system (Goeddel et al.,Nucl. Acids Res. 8:4057, 1980; and EP-A-36776) and tac promoter(Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, p. 412, 1982). A particularly useful prokaryotic host cellexpression system employs a phage λP_(L) promoter and a cI857tsthermolabile repressor sequence. Plasmid vectors available from theAmerican Type Culture Collection which incorporate derivatives of theλP_(L) promoter include plasmid pHUB2 (resident in E. coli strain JMB9,ATCC 37092) and pPLc28 (resident in E. coli RR1, ATCC 53082).

[0117] Yeast Systems

[0118] Alternatively, the polypeptides may be expressed in yeast hostcells, preferably from the Saccharomyces genus (e.g., S. cerevisiae).Other genera of yeast, such as Pichia or Kluyveromyces, may also beemployed. Yeast vectors will often contain an origin of replicationsequence from a 2μ yeast plasmid, an autonomously replicating sequence(ARS), a promoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene. Suitablepromoter sequences for yeast vectors include, among others, promotersfor metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J.Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900,1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phospho-glucose isomerase, andglucokinase. Other suitable vectors and promoters for use in yeastexpression are further described in Hitzeman, EPA-73,657. Anotheralternative is the glucose-repressible ADH2 promoter described byRussell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al. (Nature300:724, 1982). Shuttle vectors replicable in both yeast and E. coli maybe constructed by inserting DNA sequences from pBR322 for selection andreplication in E. coli (Amp^(r) gene and origin of replication) into theabove-described yeast vectors.

[0119] The yeast α-factor leader sequence may be employed to directsecretion of the polypeptide. The α-factor leader sequence is ofteninserted between the promoter sequence and the structural gene sequence.See, e.g., Kurjan et al., Cell 30:933, 1982 and Bitter et al., Proc.Natl. Acad. Sci. USA 81:5330, 1984. Other leader sequences suitable forfacilitating secretion of recombinant polypeptides from yeast hosts areknown to those of skill in the art. A leader sequence may be modifiednear its 3′ end to contain one or more restriction sites. This willfacilitate fusion of the leader sequence to the structural gene.

[0120] Yeast transformation protocols are known to those of skill in theart. One such protocol is described by Hinnen et al., Proc. Natl. Acad.Sci. USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 mg/ml adenine and 20 mg/ml uracil.

[0121] Yeast host cells transformed by vectors containing an ADH2promoter sequence may be grown for inducing expression in a “rich”medium. An example of a rich medium is one consisting of 1% yeastextract, 2% peptone, and 1% glucose supplemented with 80 mg/ml adenineand 80 mg/ml uracil. Derepression of the ADH2 promoter occurs whenglucose is exhausted from the medium.

[0122] Mammalian or Insect Systems

[0123] Mammalian or insect host cell culture systems also may beemployed to express recombinant polypeptides. Bacculovirus systems forproduction of heterologous proteins in insect cells are reviewed byLuckow and Summers, Bio/Technology 6:47 (1988). Established cell linesof mammalian origin also may be employed. Examples of suitable mammalianhost cell lines include the COS-7 line of monkey kidney cells (ATCC CRL1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, andBHK (ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived fromthe African green monkey kidney cell line CV1 (ATCC CCL 70) as describedby McMahan et al. (EMBO J. 10: 2821, 1991).

[0124] Established methods for introducing DNA into mammalian cells havebeen described (Kaufman, R. J., Large Scale Mammalian Cell Culture,1990, pp. 15-69). Additional protocols using commercially availablereagents, such as Lipofectamine lipid reagent (Gibco/BRL) orLipofectamine-Plus lipid reagent, can be used to transfect cells(Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1987). Inaddition, electroporation can be used to transfect mammalian cells usingconventional procedures, such as those in Sambrook et al. (MolecularCloning: A Laboratory Manual, 2 ed. Vol. 1-3, Cold Spring HarborLaboratory Press, 1989). Selection of stable transformants can beperformed using methods known in the art, such as, for example,resistance to cytotoxic drugs. Kaufman et al., Meth. in Enzymology185:487-511, 1990, describes several selection schemes, such asdihydrofolate reductase (DHFR) resistance. A suitable host strain forDHFR selection can be CHO strain DX-B11, which is deficient in DHFR(Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77:4216-4220, 1980). Aplasmid expressing the DHFR cDNA can be introduced into strain DX-B11,and only cells that contain the plasmid can grow in the appropriateselective media. Other examples of selectable markers that can beincorporated into an expression vector include cDNAs conferringresistance to antibiotics, such as G418 and hygromycin B. Cellsharboring the vector can be selected on the basis of resistance to thesecompounds.

[0125] Transcriptional and translational control sequences for mammalianhost cell expression vectors can be excised from viral genomes. Commonlyused promoter sequences and enhancer sequences are derived from polyomavirus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites can be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment, which can also contain a viral origin ofreplication (Fiers et al., Nature 273:113, 1978; Kaufman, Meth. inEnzymology, 1990). Smaller or larger SV40 fragments can also be used,provided the approximately 250 bp sequence extending from the Hind IIIsite toward the Bgl I site located in the SV40 viral origin ofreplication site is included.

[0126] Additional control sequences shown to improve expression ofheterologous genes from mammalian expression vectors include suchelements as the expression augmenting sequence element (EASE) derivedfrom CHO cells (Morris et al., Animal Cell Technology, 1997, pp. 529-534and PCT Application WO 97/25420) and the tripartite leader (TPL) and VAgene RNAs from Adenovirus 2 (Gingeras et al., J. Biol. Chem.257:13475-13491, 1982). The internal ribosome entry site (IRES)sequences of viral origin allows dicistronic mRNAs to be translatedefficiently (Oh and Sarnow, Current Opinion in Genetics and Development3:295-300, 1993; Ramesh et al., Nucleic Acids Research 24:2697-2700,1996). Expression of a heterologous cDNA as part of a dicistronic mRNAfollowed by the gene for a selectable marker (e.g. DHFR) has been shownto improve transfectability of the host and expression of theheterologous cDNA (Kaufman, Meth. in Enzymology, 1990). Exemplaryexpression vectors that employ dicistronic mRNAs are pTR-DC/GFPdescribed by Mosser et al., Biotechniques 22:150-161, 1997, and p2A5Idescribed by Morris et al., Animal Cell Technology, 1997, pp. 529-534.

[0127] A useful high expression vector, pCAVNOT, has been described byMosley et al., Cell 59:335-348, 1989. Other expression vectors for usein mammalian host cells can be constructed as disclosed by Okayama andBerg (Mol. Cell. Biol. 3:280, 1983). A useful system for stable highlevel expression of mammalian cDNAs in C127 murine mammary epithelialcells can be constructed substantially as described by Cosman et al.(Mol. Immunol. 23:935, 1986). A useful high expression vector, PMLSVN1/N4, described by Cosman et al., Nature 312:768, 1984, has beendeposited as ATCC 39890. Additional useful mammalian expression vectorsare described in EP-A-0367566, and in WO 91/18982, incorporated byreference herein. In yet another alternative, the vectors can be derivedfrom retroviruses.

[0128] Another useful expression vector, pFLAG⁷, can be used. FLAG⁷technology is centered on the fusion of a low molecular weight (1 kD),hydrophilic, FLAG⁷ marker peptide to the N-terminus of a recombinantprotein expressed by pFLAG⁷ expression vectors.

[0129] Regarding signal peptides that may be employed, the native signalpeptide may be replaced by a heterologous signal peptide or leadersequence, if desired. The choice of signal peptide or leader may dependon factors such as the type of host cells in which the recombinantpolypeptide is to be produced. To illustrate, examples of heterologoussignal peptides that are functional in mammalian host cells include thesignal sequence for interleukin-7 (IL-7) described in U.S. Pat. No.4,965,195; the signal sequence for interleukin-2 receptor described inCosman et al., Nature 312:768 (1984); the interleukin-4 receptor signalpeptide described in EP 367,566; the type I interleukin-1 receptorsignal peptide described in U.S. Pat. No. 4,968,607; and the type IIinterleukin-1 receptor signal peptide described in EP 460,846.

[0130] Purification

[0131] The invention also includes methods of isolating and purifyingthe polypeptides and fragments thereof.

[0132] Isolation and Purification

[0133] The “isolated” polypeptides or fragments thereof encompassed bythis invention are polypeptides or fragments that are not in anenvironment identical to an environment in which it or they can be foundin nature. The “purified” polypeptides or fragments thereof encompassedby this invention are essentially free of association with otherproteins or polypeptides, for example, as a purification product ofrecombinant expression systems such as those described above or as apurified product from a non-recombinant source such as naturallyoccurring cells and/or tissues.

[0134] In one preferred embodiment, the purification of recombinantpolypeptides or fragments can be accomplished using fusions ofpolypeptides or fragments of the invention to another polypeptide to aidin the purification of polypeptides or fragments of the invention. Suchfusion partners can include the poly-His or other antigenicidentification peptides described above as well as the Fc moietiesdescribed previously.

[0135] With respect to any type of host cell, as is known to the skilledartisan, procedures for purifying a recombinant polypeptide or fragmentwill vary according to such factors as the type of host cells employedand whether or not the recombinant polypeptide or fragment is secretedinto the culture medium.

[0136] In general, the recombinant polypeptide or fragment can beisolated from the host cells if not secreted, or from the medium orsupernatant if soluble and secreted, followed by one or moreconcentration, salting-out, ion exchange, hydrophobic interaction,affinity purification or size exclusion chromatography steps. As tospecific ways to accomplish these steps, the culture medium first can beconcentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. Following the concentration step, the concentrate can be appliedto a purification matrix such as a gel filtration medium. Alternatively,an anion exchange resin can be employed, for example, a matrix orsubstrate having pendant diethylaminoethyl (DEAE) groups. The matricescan be acrylamide, agarose, dextran, cellulose or other types commonlyemployed in protein purification. Alternatively, a cation exchange stepcan be employed. Suitable cation exchangers include various insolublematrices comprising sulfopropyl or carboxymethyl groups. In addition, achromatofocusing step can be employed. Alternatively, a hydrophobicinteraction chromatography step can be employed. Suitable matrices canbe phenyl or octyl moieties bound to resins. In addition, affinitychromatography with a matrix which selectively binds the recombinantprotein can be employed. Examples of such resins employed are lectincolumns, dye columns, and metal-chelating columns. Finally, one or morereverse-phase high performance liquid chromatography (RP-HPLC) stepsemploying hydrophobic RP-HPLC media, (e.g., silica gel or polymer resinhaving pendant methyl, octyl, octyldecyl or other aliphatic groups) canbe employed to further purify the polypeptides. Some or all of theforegoing purification steps, in various combinations, are well knownand can be employed to provide an isolated and purified recombinantprotein.

[0137] It is also possible to utilize an affinity column comprising apolypeptide-binding protein of the invention, such as a monoclonalantibody generated against polypeptides of the invention, toaffinity-purify expressed polypeptides. These polypeptides can beremoved from an affinity column using conventional techniques, e.g., ina high salt elution buffer and then dialyzed into a lower salt bufferfor use or by changing pH or other components depending on the affinitymatrix utilized, or be competitively removed using the naturallyoccurring substrate of the affinity moiety, such as a polypeptidederived from the invention.

[0138] In this aspect of the invention, polypeptide-binding proteins,such as the anti-polypeptide antibodies of the invention or otherproteins that may interact with the polypeptide of the invention, can bebound to a solid phase support such as a column chromatography matrix ora similar substrate suitable for identifying, separating, or purifyingcells that express polypeptides of the invention on their surface.Adherence of polypeptide-binding proteins of the invention to a solidphase contacting surface can be accomplished by any means. For example,magnetic microspheres can be coated with these polypeptide-bindingproteins and held in the incubation vessel through a magnetic field.Suspensions of cell mixtures are contacted with the solid phase that hassuch polypeptide-binding proteins thereon. Cells having polypeptides ofthe invention on their surface bind to the fixed polypeptide-bindingprotein and unbound cells then are washed away. This affinity-bindingmethod is useful for purifying, screening, or separating suchpolypeptide-expressing cells from solution. Methods of releasingpositively selected cells from the solid phase are known in the art andencompass, for example, the use of enzymes. Such enzymes are preferablynon-toxic and non-injurious to the cells and are preferably directed tocleaving the cell-surface binding partner.

[0139] Alternatively, mixtures of cells suspected of containingpolypeptide-expressing cells of the invention first can be incubatedwith a biotinylated polypeptide-binding protein of the invention.Incubation periods are typically at least one hour in duration to ensuresufficient binding to polypeptides of the invention. The resultingmixture then is passed through a column packed with avidin-coated beads,whereby the high affinity of biotin for avidin provides the binding ofthe polypeptide-binding cells to the beads. Use of avidin-coated beadsis known in the art. See Berenson, et al. J. Cell. Biochem., 10D:239(1986). Wash of unbound material and the release of the bound cells isperformed using conventional methods.

[0140] The desired degree of purity depends on the intended use of theprotein. A relatively high degree of purity is desired when thepolypeptide is to be administered in vivo, for example. In such a case,the polypeptides are purified such that no protein bands correspondingto other proteins are detectable upon analysis by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE). It will be recognized by one skilled in thepertinent field that multiple bands corresponding to the polypeptide maybe visualized by SDS-PAGE, due to differential glycosylation,differential post-translational processing, and the like. Mostpreferably, the polypeptide of the invention is purified to substantialhomogeneity, as indicated by a single protein band upon analysis bySDS-PAGE. The protein band may be visualized by silver staining,Coomassie blue staining, or (if the protein is radiolabeled) byautoradiography.

[0141] Assays

[0142] The purified polypeptides of the invention (including proteins,polypeptides, fragments, variants, oligomers, and other forms) may betested for the ability to bind the binding partner in any suitableassay, such as a conventional binding assay. To illustrate, thepolypeptide may be labeled with a detectable reagent (e.g., aradionuclide, chromophore, enzyme that catalyzes a colorimetric orfluorometric reaction, and the like). The labeled polypeptide iscontacted with cells expressing the binding partner. The cells then arewashed to remove unbound labeled polypeptide, and the presence ofcell-bound label is determined by a suitable technique, chosen accordingto the nature of the label.

[0143] One example of a binding assay procedure is as follows. Arecombinant expression vector containing the binding partner cDNA isconstructed using methods well known in the art. CV1-EBNA-1 cells in 10cm² dishes are transfected with the recombinant expression vector.CV-1/EBNA-1 cells (ATCC CRL 10478) constitutively express EBV nuclearantigen-1 driven from the CMV immediate-early enhancer/promoter.CV1-EBNA-1 was derived from the African Green Monkey kidney cell lineCV-1 (ATCC CCL 70), as described by McMahan et al. (EMBO J. 10:2821,1991).

[0144] The transfected cells are cultured for 24 hours, and the cells ineach dish then are split into a 24-well plate. After culturing anadditional 48 hours, the transfected cells (about 4×10⁴ cells/well) arewashed with BM-NFDM, which is binding medium (RPMI 1640 containing 25mg/ml bovine serum albumin, 2 mg/ml sodium azide, 20 mM Hepes pH 7.2) towhich 50 mg/ml nonfat dry milk has been added. The cells then areincubated for 1 hour at 37° C. with various concentrations of, forexample, a soluble polypeptide/Fc fusion protein made as set forthabove. Cells then are washed and incubated with a constant saturatingconcentration of a ¹²⁵I-mouse anti-human IgG in binding medium, withgentle agitation for 1 hour at 37° C. After extensive washing, cells arereleased via trypsinization.

[0145] The mouse anti-human IgG employed above is directed against theFc region of human IgG and can be obtained from Jackson ImmunoresearchLaboratories, Inc., West Grove, Pa. The antibody is radioiodinated usingthe standard chloramine-T method. The antibody will bind to the Fcportion of any polypeptide/Fc protein that has bound to the cells. Inall assays, non-specific binding of ¹²⁵I-antibody is assayed in theabsence of the Fc fusion protein/Fc, as well as in the presence of theFc fusion protein and a 200-fold molar excess of unlabeled mouseanti-human IgG antibody.

[0146] Cell-bound ¹²⁵I-antibody is quantified on a Packard Autogammacounter. Affinity calculations (Scatchard, Ann. N.Y. Acad. Sci. 51:660,1949) are generated on RS/1 (BBN Software, Boston, Mass.) run on aMicrovax computer.

[0147] Another type of suitable binding assay is a competitive bindingassay. To illustrate, biological activity of a variant may be determinedby assaying for the variant's ability to compete with the native proteinfor binding to the binding partner.

[0148] Competitive binding assays can be performed by conventionalmethodology. Reagents that may be employed in competitive binding assaysinclude radiolabeled polypeptides of the invention and intact cellsexpressing the binding partner (endogenous or recombinant). For example,a radiolabeled soluble IL-1 zeta fragment can be used to compete with asoluble IL-1 zeta variant for binding to cell surface IL-1 zetareceptors. Instead of intact cells, one could substitute a solublebinding partner/Fc fusion protein bound to a solid phase through theinteraction of Protein A or Protein G (on the solid phase) with the Fcmoiety. Chromatography columns that contain Protein A and Protein Ginclude those available from Pharmacia Biotech, Inc., Piscataway, N.J.

[0149] Another type of competitive binding assay utilizes radiolabeledsoluble binding partner, such as a soluble IL-1 zeta receptor/Fc fusionor Xrec2 ligand/Fc fusion protein, and intact cells expressing thebinding partner. Qualitative results can be obtained by competitiveautoradiographic plate binding assays, while Scatchard plots (Scatchard,Ann. N.Y. Acad. Sci. 51:660, 1949) may be utilized to generatequantitative results.

[0150] Use of IL-1 ZETA, TDZ.1, TDZ.2. TDZ.3 AND Xrec2 Nucleic Acid orOligonucleotides

[0151] In addition to being used to express polypeptides as describedabove, the nucleic acids of the invention, including DNA, andoligonucleotides thereof can be used:

[0152] as probes to identify nucleic acid encoding proteins of the IL-1ligand and receptor families;

[0153] to identify human chromosomes 2 and X;

[0154] to map genes on human chromosomes 2 and X;

[0155] to identify genes associated with certain diseases, syndromes, orother conditions associated with human chromosomes 2 and X;

[0156] as single-stranded sense or antisense oligonucleotides, toinhibit expression of polypeptides encoded by the IL-1 zeta, TDZ.1,TDZ.2, TDZ.3 and Xrec2 genes;

[0157] to help detect defective genes in an individual; and

[0158] for gene therapy.

[0159] Probes

[0160] Among the uses of nucleic acids of the invention is the use offragments as probes or primers. Such fragments generally comprise atleast about 17 contiguous nucleotides of a DNA sequence. In otherembodiments, a DNA fragment comprises at least 30, or at least 60,contiguous nucleotides of a DNA sequence.

[0161] Because homologs of SEQ ID NOs:1, 2, 5, 6 and 7, from othermammalian species, are contemplated herein, probes based on the humanDNA sequences of SEQ ID NOs: 1, 2, 5, 6 and 7 may be used to screen cDNAlibraries derived from other mammalian species, using conventionalcross-species hybridization techniques.

[0162] Using knowledge of the genetic code in combination with the aminoacid sequences set forth above, sets of degenerate oligonucleotides canbe prepared. Such oligonucleotides are useful as primers, e.g., inpolymerase chain reactions (PCR), whereby DNA fragments are isolated andamplified.

[0163] Chromosome Mapping

[0164] All or a portion of the nucleic acids of IL-1 zeta of SEQ ID NO:1and of Xrec2 of SEQ ID NO:2, including oligonucleotides, can be used bythose skilled in the art using well-known techniques to identify thehuman chromosomes 2 and X, respectively, as well as the specific locusthereof, that contains the DNA of IL-1 ligand and IL-1 receptor familymembers. Useful techniques include, but are not limited to, using thesequence or portions, including oligonucleotides, as a probe in variouswell-known techniques such as radiation hybrid mapping (highresolution), in situ hybridization to chromosome spreads (moderateresolution), and Southern blot hybridization to hybrid cell linescontaining individual human chromosomes (low resolution).

[0165] For example, chromosomes can be mapped by radiationhybridization. PCR is performed using the Whitehead Institute/MIT Centerfor Genome Research Genebridge4 panel of 93 radiation hybrids(http://www-genome.wi.mit.edu/ftp/distribution/human_STS_releases/july97/rhmap/genebridge4.html).Primers are used which lie within a putative exon of the gene ofinterest and which amplify a product from human genomic DNA, but do notamplify hamster genomic DNA. The results of the PCRs are converted intoa data vector that is submitted to the Whitehead/MIT Radiation Mappingsite on the internet (http://www-seq.wi.mit.edu). The data is scored andthe chromosomal assignment and placement relative to known Sequence TagSite (STS) markers on the radiation hybrid map is provided. Thefollowing web site provides additional information about radiationhybrid mapping: http://www-genome.wi.mit.edu/ftp/distribution/humanSTS_releases/july97/07-97.1NTRO.html).

[0166] Identifying Associated Diseases

[0167] As set forth below, IL-1 zeta of SEQ ID NO:1, IL-1 zeta splicevariants, and Xrec2 of SEQ ID NO:2 have been mapped by radiationhybridization and high-throughput-shotgun sequencing to the 2q11-12 andXp22 regions of human chromosomes 2 and X, respectively. Humanchromosome 2 is associated with specific diseases which include but arenot limited to glaucoma, ectodermal dysplasia, insulin-dependentdiabetes mellitus, wrinkly skin syndrome, T-cell leukemia/lymphoma, andtibial muscular dystrophy while human chromosome X is associated withretinoschisis, lissencephaly, subcortical laminalheteropia, mentalretardation, cowchock syndrome, bazex syndrome, hypertrichosis,lymphoproliferative syndrome, immunodeficiency, Langer mesomelicdysplasia, and leukemia. Thus, the nucleic acids of SEQ ID NOs:1 and 2or a fragment thereof can be used by one skilled in the art usingwell-known techniques to analyze abnormalities associated with genemapping to chromosomes 2 and X. This enables one to distinguishconditions in which this marker is rearranged or deleted. In addition,nucleotides of SEQ ID NOs: 1, 2, 5, 6 and 7 or a fragment thereof can beused as a positional marker to map other genes of unknown location.

[0168] The DNA may be used in developing treatments for any disordermediated (directly or indirectly) by defective, or insufficient amountsof, the genes corresponding to the nucleic acids of the invention.Disclosure herein of native nucleotide sequences permits the detectionof defective genes, and the replacement thereof with normal genes.Defective genes may be detected in in vitro diagnostic assays, and bycomparison of a native nucleotide sequence disclosed herein with that ofa gene derived from a person suspected of harboring a defect in thisgene.

[0169] Sense-Antisense

[0170] Other useful fragments of the nucleic acids include antisense orsense oligonucleotides comprising a single-stranded nucleic acidsequence (either RNA or DNA) capable of binding to target mRNA (sense)or DNA (antisense) sequences. Antisense or sense oligonucleotidesaccording to the present invention comprise a fragment of DNA (SEQ IDNOs: 1, 2, 5, 6 and 7). Such a fragment generally comprises at leastabout 14 nucleotides, preferably from about 14 to about 30 nucleotides.The ability to derive an antisense or a sense oligonucleotide, basedupon a cDNA sequence encoding a given protein is described in, forexample, Stein and Cohen (Cancer Res. 48:2659, 1988) and van der Krol etal. (BioTechniques 6:958, 1988).

[0171] Binding of antisense or sense oligonucleotides to target nucleicacid sequences results in the formation of duplexes that block orinhibit protein expression by one of several means, including enhanceddegradation of the mRNA by RNAseH, inhibition of splicing, prematuretermination of transcription or translation, or by other means. Theantisense oligonucleotides thus may be used to block expression ofproteins. Antisense or sense oligonucleotides further compriseoligonucleotides having modified sugar-phosphodiester backbones (orother sugar linkages, such as those described in WO91/06629) and whereinsuch sugar linkages are resistant to endogenous nucleases. Sucholigonucleotides with resistant sugar linkages are stable in vivo (i.e.,capable of resisting enzymatic degradation) but retain sequencespecificity to be able to bind to target nucleotide sequences.

[0172] Other examples of sense or antisense oligonucleotides includethose oligonucleotides which are covalently linked to organic moieties,such as those described in WO 90/10448, and other moieties thatincreases affinity of the oligonucleotide for a target nucleic acidsequence, such as poly-(L-lysine). Further still, intercalating agents,such as ellipticine, and alkylating agents or metal complexes may beattached to sense or antisense oligonucleotides to modify bindingspecificities of the antisense or sense oligonucleotide for the targetnucleotide sequence.

[0173] Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, lipofection, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus.

[0174] Sense or antisense oligonucleotides also may be introduced into acell containing the target nucleotide sequence by formation of aconjugate with a ligand binding molecule, as described in WO 91/04753.Suitable ligand binding molecules include, but are not limited to, cellsurface receptors, growth factors, other cytokines, or other ligandsthat bind to cell surface receptors. Preferably, conjugation of theligand binding molecule does not substantially interfere with theability of the ligand binding molecule to bind to its correspondingmolecule or receptor, or block entry of the sense or antisenseoligonucleotide or its conjugated version into the cell.

[0175] Alternatively, a sense or an antisense oligonucleotide may beintroduced into a cell containing the target nucleic acid sequence byformation of an oligonucleotide-lipid complex, as described in WO90/10448. The sense or antisense oligonucleotide-lipid complex ispreferably dissociated within the cell by an endogenous lipase.

[0176] IL-1 zeta anti-sense are useful as therapeutics to treat medicalconditions and disease associated with immune system dysfunction andIL-12 production. Such medical conditions and disease are describedbelow and include the deleterious effects of inflammation andauto-immune diseases. Accordingly, IL-1 zeta anti-sense are IL-12antagonists and are useful in treating disease and medical conditionsthat are benefited by IL-12 expression downregulation.

[0177] Use of IL-1 Zeta, TDZ.1, TDZ.2 TDZ.3 and Xrec2 Polypeptides andFragmented Polypeptides

[0178] Uses include, but are not limited to, the following:

[0179] Purifying proteins and measuring activity thereof

[0180] Delivery Agents

[0181] Therapeutic and Research Reagents

[0182] Molecular weight and Isoelectric focusing markers

[0183] Controls for peptide fragmentation

[0184] Identification of unknown proteins

[0185] Preparation of Antibodies

[0186] Purification Reagents

[0187] Each of the polypeptides of the invention finds use as a proteinpurification reagent. The polypeptides may be attached to a solidsupport material and used to purify the binding partner proteins byaffinity chromatography. In particular embodiments, a polypeptide (inany form described herein that is capable of binding the bindingpartner) is attached to a solid support by conventional procedures. Asone example, chromatography columns containing functional groups thatwill react with functional groups on amino acid side chains of proteinsare available (Pharmacia Biotech, Inc., Piscataway, N.J.). In analternative, a polypeptide/Fc protein (as discussed above) is attachedto Protein A- or Protein G-containing chromatography columns throughinteraction with the Fc moiety.

[0188] The polypeptide also finds use in purifying or identifying cellsthat express the binding partner on the cell surface. Polypeptides arebound to a solid phase such as a column chromatography matrix or asimilar suitable substrate. For example, magnetic microspheres can becoated with the polypeptides and held in an incubation vessel through amagnetic field. Suspensions of cell mixtures containing the bindingpartner expressing cells are contacted with the solid phase having thepolypeptides thereon. Cells expressing the binding partner on the cellsurface bind to the fixed polypeptides, and unbound cells then arewashed away.

[0189] Alternatively, the polypeptides can be conjugated to a detectablemoiety, then incubated with cells to be tested for binding partnerexpression. After incubation, unbound labeled matter is removed and thepresence or absence of the detectable moiety on the cells is determined.

[0190] In a further alternative, mixtures of cells suspected ofcontaining cells expressing the binding partner are incubated withbiotinylated polypeptides. Incubation periods are typically at least onehour in duration to ensure sufficient binding. The resulting mixturethen is passed through a column packed with avidin-coated beads, wherebythe high affinity of biotin for avidin provides binding of the desiredcells to the beads. Procedures for using avidin-coated beads are known(see Berenson, et al. J. Cell. Biochem., 10D:239, 1986). Washing toremove unbound material, and the release of the bound cells, areperformed using conventional methods.

[0191] Measuring Activity

[0192] Polypeptides also find use in measuring the biological activityof the binding partner protein in terms of their binding affinity. Thepolypeptides thus may be employed by those conducting “qualityassurance” studies, e.g., to monitor shelf life and stability of proteinunder different conditions. For example, the polypeptides may beemployed in a binding affinity study to measure the biological activityof a binding partner protein that has been stored at differenttemperatures, or produced in different cell types. The proteins also maybe used to determine whether biological activity is retained aftermodification of a binding partner protein (e.g., chemical modification,truncation, mutation, etc.). The binding affinity of the modifiedbinding partner protein is compared to that of an unmodified bindingpartner protein to detect any adverse impact of the modifications onbiological activity of the binding partner. The biological activity of abinding partner protein thus can be ascertained before it is used in aresearch study, for example.

[0193] Delivery Agents

[0194] The polypeptides also find use as carriers for delivering agentsattached thereto to cells bearing the binding partner. The polypeptidesthus can be used to deliver diagnostic or therapeutic agents to suchcells (or to other cell types found to express the binding partner onthe cell surface) in in vitro or in vivo procedures.

[0195] Detectable (diagnostic) and therapeutic agents that may beattached to a polypeptide include, but are not limited to, toxins, othercytotoxic agents, drugs, radionuclides, chromophores, enzymes thatcatalyze a calorimetric or fluorometric reaction, and the like, with theparticular agent being chosen according to the intended application.Among the toxins are ricin, abrin, diphtheria toxin, Pseudomonasaeruginosa exotoxin A, ribosomal inactivating proteins, mycotoxins suchas trichothecenes, and derivatives and fragments (e.g., single chains)thereof. Radionuclides suitable for diagnostic use include, but are notlimited to, ¹²³I, ¹³¹I, ^(99m)Tc, ¹¹¹In, and ⁷⁶Br. Examples ofradionuclides suitable for therapeutic use are ¹³¹I, ²¹¹At, ⁷⁷Br, ¹⁸⁶Re,¹⁸⁸Re, ²¹²Pb, ²¹²Bi, ¹⁰⁹Pd, ⁶⁴Cu, and ⁶⁷Cu.

[0196] Such agents may be attached to the polypeptide by any suitableconventional procedure. The polypeptide comprises functional groups onamino acid side chains that can be reacted with functional groups on adesired agent to form covalent bonds, for example. Alternatively, theprotein or agent may be derivatized to generate or attach a desiredreactive functional group. The derivatization may involve attachment ofone of the bifunctional coupling reagents available for attachingvarious molecules to proteins (Pierce Chemical Company, Rockford, Ill.).A number of techniques for radiolabeling proteins are known.Radionuclide metals may be attached to polypeptides by using a suitablebifunctional chelating agent, for example.

[0197] Conjugates comprising polypeptides and a suitable diagnostic ortherapeutic agent (preferably covalently linked) are thus prepared. Theconjugates are administered or otherwise employed in an amountappropriate for the particular application.

[0198] Therapeutic Agents

[0199] Polypeptides of the invention may be used in developingtreatments for any disorder mediated (directly or indirectly) bydefective, or insufficient amounts of the polypeptides. Thesepolypeptides may be administered to a mammal afflicted with such adisorder.

[0200] The polypeptides may also be employed in inhibiting a biologicalactivity of the binding partner, in in vitro or in vivo procedures. Forexample, a purified Xrec2 receptor polypeptide may be used to inhibitbinding of Xrec2 ligand to endogenous cell surface Xrec2 receptor, or apurified IL-1 zeta polypeptide, or any of its splice variants can beused to inhibit binding of endogenous IL-1 zeta or splice variants toits cell surface receptor. Biological effects that result from thebinding of Xrec2 ligand to endogenous Xrec2 receptors thus areinhibited. In particular, IL-1 zeta polypeptides and fragments of thesepolypeptides that induce IL-12 expression are useful to upregulate IL-12expression in individuals who can benefit from increased IL-12production, including individuals who benefit from enhanced cellmediated immunity. Diseases and medical conditions treatable withagonists of IL-1 zeta polypeptide, as described below, may be suitablytreated using IL-1 zeta polypeptides and fragments of this invention.

[0201] Polypeptides of the invention may be administered to a mammal totreat a binding partner-mediated disorder. Such binding partner-mediateddisorders include conditions caused (directly or indirectly) orexacerbated by the binding partner.

[0202] Compositions of the present invention may contain a polypeptidein any form described herein, such as native proteins, variants,derivatives, oligomers, and biologically active fragments. In particularembodiments, the composition comprises a soluble polypeptide or anoligomer comprising soluble polypeptides of the invention.

[0203] Compositions comprising an effective amount of a polypeptide ofthe present invention, in combination with other components such as aphysiologically acceptable diluent, carrier, or excipient, are providedherein. The polypeptides can be formulated according to known methodsused to prepare pharmaceutically useful compositions. They can becombined in admixture, either as the sole active material or with otherknown active materials suitable for a given indication, withpharmaceutically acceptable diluents (e.g., saline, Tris-HCl, acetate,and phosphate buffered solutions), preservatives (e.g., thimerosal,benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/orcarriers. Suitable formulations for pharmaceutical compositions includethose described in Remington's Pharmaceutical Sciences, 16th ed. 1980,Mack Publishing Company, Easton, Pa.

[0204] In addition, such compositions can be complexed with polyethyleneglycol (PEG), metal ions, or incorporated into polymeric compounds suchas polyacetic acid, polyglycolic acid, hydrogels, dextran, etc., orincorporated into liposomes, microemulsions, micelles, unilamellar ormultilamellar vesicles, erythrocyte ghosts or spheroblasts. Suchcompositions will influence the physical state, solubility, stability,rate of in vivo release, and rate of in vivo clearance, and are thuschosen according to the intended application.

[0205] The compositions of the invention can be administered in anysuitable manner, e.g., topically, parenterally, or by inhalation. Theterm “parenteral” includes injection, e.g., by subcutaneous,intravenous, or intramuscular routes, also including localizedadministration, e.g., at a site of disease or injury. Sustained releasefrom implants is also contemplated. One skilled in the pertinent artwill recognize that suitable dosages will vary, depending upon suchfactors as the nature of the disorder to be treated, the patient's bodyweight, age, and general condition, and the route of administration.Preliminary doses can be determined according to animal tests, and thescaling of dosages for human administration is performed according toart-accepted practices.

[0206] Compositions comprising nucleic acids in physiologicallyacceptable formulations are also contemplated. DNA may be formulated forinjection, for example.

[0207] Research Agents

[0208] Another use of the polypeptide of the present invention is as aresearch tool for studying the biological effects that result from theinteractions of IL-1 zeta, or any of its splice variants, with itsbinding partner, and of Xrec2 with its binding partner, or frominhibiting these interactions, on different cell types. Polypeptidesalso may be employed in in vitro assays for detecting IL-1 zeta, Xrec2,the respective binding partners or the interactions thereof.

[0209] Another embodiment of the invention relates to uses of thepolypeptides of the invention to study cell signal transduction. IL-1family ligands and receptors play a central role in protection againstinfection and immune inflammatory responses which includes cellularsignal transduction, activating vascular endothelial cells andlymphocytes, induction of inflammatory cytokines, acute phase proteins,hematopoiesis, fever, bone resorption, prostaglandins,metalloproteinases, and adhesion molecules. With the continued increasein the number of known IL-1 family members, a suitable classificationscheme is one based on comparing polypeptide structure as well asfunction (activation and regulatory properties). Thus, IL-1 zeta, TDZ.1,TDZ.2, and TDZ.3, like other IL-1 family ligands (IL-1α, IL-1β, andIL-18) and Xrec2, like other IL-1R family receptors (IL-1RI, IL-1RII,IL-1Rrp1, and AcPL), would likely be involved in many of the functionsnoted above as well as promote inflammatory responses and thereforeperhaps be involved in the causation and maintenance of inflammatoryand/or autoimmune diseases such as rheumatoid arthritis, inflammatorybowel disease, and psoriasis. As such, alterations in the expressionand/or activation of the polypeptides of the invention can have profoundeffects on a plethora of cellular processes, including, but not limitedto, activation or inhibition of cell specific responses andproliferation. Expression of cloned IL-1 zeta, Xrec2, or of functionallyinactive mutants thereof can be used to identify the role a particularprotein plays in mediating specific signaling events.

[0210] IL-1 mediated cellular signaling often involves a molecularactivation cascade, during which a receptor propagates a ligand-receptormediated signal by specifically activating intracellular kinases whichphosphorylate target substrates. These substrates can themselves bekinases which become activated following phosphorylation. Alternatively,they can be adapter molecules that facilitate down stream signalingthrough protein-protein interaction following phosphorylation.Regardless of the nature of the substrate molecule(s), expressedfunctionally active versions of Xrec2, IL-1 zeta, its splice variants,and their binding partners can be used to identify what substrate(s)were recognized and activated by the polypeptides of the invention. Assuch, these novel polypeptides can be used as reagents to identify novelmolecules involved in signal transduction pathways.

[0211] Molecular Weight, Isoelectric Point Markers

[0212] The polypeptides of the present invention can be subjected tofragmentation into smaller peptides by chemical and enzymatic means, andthe peptide fragments so produced can be used in the analysis of otherproteins or polypeptides. For example, such peptide fragments can beused as peptide molecular weight markers, peptide isoelectric pointmarkers, or in the analysis of the degree of peptide fragmentation.Thus, the invention also includes these polypeptides and peptidefragments, as well as kits to aid in the determination of the apparentmolecular weight and isoelectric point of an unknown protein and kits toassess the degree of fragmentation of an unknown protein.

[0213] Although all methods of fragmentation are encompassed by theinvention, chemical fragmentation is a preferred embodiment, andincludes the use of cyanogen bromide to cleave under neutral or acidicconditions such that specific cleavage occurs at methionine residues (E.Gross, Methods in Enz. 11:238-255, 1967). This can further includeadditional steps, such as a carboxymethylation step to convert cysteineresidues to an unreactive species.

[0214] Enzymatic fragmentation is another preferred embodiment, andincludes the use of a protease such as Asparaginylendo-peptidase,Arginylendo-peptidase, Achromobacter protease I, Trypsin, Staphlococcusaureus V8 protease, Endoproteinase Asp-N, or Endoproteinase Lys-C underconventional conditions to result in cleavage at specific amino acidresidues. Asparaginylendo-peptidase can cleave specifically on thecarboxyl side of the asparagine residues present within the polypeptidesof the invention. Arginylendo-peptidase can cleave specifically on thecarboxyl side of the arginine residues present within thesepolypeptides. Achromobacter protease I can cleave specifically on thecarboxyl side of the lysine residues present within the polypeptides(Sakiyama and Nakat, U.S. Pat. No. 5,248,599; T. Masaki et al., Biochim.Biophys. Acta 660:44-50, 1981; T. Masaki et al., Biochim. Biophys. Acta660:51-55, 1981). Trypsin can cleave specifically on the carboxyl sideof the arginine and lysine residues present within polypeptides of theinvention. Enzymatic fragmentation may also occur with a protease thatcleaves at multiple amino acid residues. For example, Staphlococcusaureus V8 protease can cleave specifically on the carboxyl side of theaspartic and glutamic acid residues present within polypeptides (D. W.Cleveland, J. Biol. Chem. 3:1102-1106, 1977). Endoproteinase Asp-N cancleave specifically on the amino side of the asparagine residues presentwithin polypeptides. Endoproteinase Lys-C can cleave specifically on thecarboxyl side of the lysine residues present within polypeptides of theinvention. Other enzymatic and chemical treatments can likewise be usedto specifically fragment these polypeptides into a unique set ofspecific peptides.

[0215] Of course, the peptides and fragments of the polypeptides of theinvention can also be produced by conventional recombinant processes andsynthetic processes well known in the art. With regard to recombinantprocesses, the polypeptides and peptide fragments encompassed byinvention can have variable molecular weights, depending upon the hostcell in which they are expressed. Glycosylation of polypeptides andpeptide fragments of the invention in various cell types can result invariations of the molecular weight of these pieces, depending upon theextent of modification. The size of these pieces can be mostheterogeneous with fragments of polypeptide derived from theextracellular portion of the polypeptide. Consistent polypeptides andpeptide fragments can be obtained by using polypeptides derived entirelyfrom the transmembrane and cytoplasmic regions, pretreating withN-glycanase to remove glycosylation, or expressing the polypeptides inbacterial hosts.

[0216] The molecular weight of these polypeptides can also be varied byfusing additional peptide sequences to both the amino and carboxylterminal ends of polypeptides of the invention. Fusions of additionalpeptide sequences at the amino and carboxyl terminal ends ofpolypeptides of the invention can be used to enhance expression of thesepolypeptides or aid in the purification of the protein. In addition,fusions of additional peptide sequences at the amino and carboxylterminal ends of polypeptides of the invention will alter some, butusually not all, of the fragmented peptides of the polypeptidesgenerated by enzymatic or chemical treatment. Of course, mutations canbe introduced into polypeptides of the invention using routine and knowntechniques of molecular biology. For example, a mutation can be designedso as to eliminate a site of proteolytic cleavage by a specific enzymeor a site of cleavage by a specific chemically induced fragmentationprocedure. The elimination of the site will alter the peptidefingerprint of polypeptides of the invention upon fragmentation with thespecific enzyme or chemical procedure.

[0217] The polypeptides and the resultant fragmented peptides can beanalyzed by methods including sedimentation, electrophoresis,chromatography, and mass spectrometry to determine their molecularweights. Because the unique amino acid sequence of each piece specifiesa molecular weight, these pieces can thereafter serve as molecularweight markers using such analysis techniques to assist in thedetermination of the molecular weight of an unknown protein,polypeptides or fragments thereof. The molecular weight markers of theinvention serve particularly well as molecular weight markers for theestimation of the apparent molecular weight of proteins that havesimilar apparent molecular weights and, consequently, allow increasedaccuracy in the determination of apparent molecular weight of proteins.

[0218] When the invention relates to the use of fragmented peptidemolecular weight markers, those markers are preferably at least 10 aminoacids in size. More preferably, these fragmented peptide molecularweight markers are between 10 and 100 amino acids in size. Even morepreferable are fragmented peptide molecular weight markers between 10and 50 amino acids in size and especially between 10 and 35 amino acidsin size. Most preferable are fragmented peptide molecular weight markersbetween 10 and 20 amino acids in size.

[0219] Among the methods for determining molecular weight aresedimentation, gel electrophoresis, chromatography, and massspectrometry. A particularly preferred embodiment is denaturingpolyacrylamide gel electrophoresis (U. K. Laemmli, Nature 227:680-685,1970). Conventionally, the method uses two separate lanes of a gelcontaining sodium dodecyl sulfate and a concentration of acrylamidebetween 6-20%. The ability to simultaneously resolve the marker and thesample under identical conditions allows for increased accuracy. It isunderstood, of course, that many different techniques can be used forthe determination of the molecular weight of an unknown protein usingpolypeptides of the invention, and that this embodiment in no way limitsthe scope of the invention.

[0220] Each unglycosylated polypeptide or fragment thereof has a p1 thatis intrinsically determined by its unique amino acid sequence (which pIcan be estimated by the skilled artisan using any of the computerprograms designed to predict pI values currently available, calculatedusing any well-known amino acid pKa table, or measured empirically).Therefore these polypeptides and fragments thereof can serve as specificmarkers to assist in the determination of the isoelectric point of anunknown protein, polypeptide, or fragmented peptide using techniquessuch as isoelectric focusing. These polypeptide or fragmented peptidemarkers serve particularly well for the estimation of apparentisoelectric points of unknown proteins that have apparent isoelectricpoints close to that of the polypeptide or fragmented peptide markers ofthe invention.

[0221] The technique of isoelectric focusing can be further combinedwith other techniques such as gel electrophoresis to simultaneouslyseparate a protein on the basis of molecular weight and charge. Theability to simultaneously resolve these polypeptide or fragmentedpeptide markers and the unknown protein under identical conditionsallows for increased accuracy in the determination of the apparentisoelectric point of the unknown protein. This is of particular interestin techniques, such as two dimensional electrophoresis (T. D. Brock andM. T. Madigan, Biology of Microorganisms 76-77 (Prentice Hall, 6d ed.1991)), where the nature of the procedure dictates that any markersshould be resolved simultaneously with the unknown protein. In addition,with such methods, these polypeptides and fragmented peptides thereofcan assist in the determination of both the isoelectric point andmolecular weight of an unknown protein or fragmented peptide.

[0222] Polypeptides and fragmented peptides can be visualized using twodifferent methods that allow a discrimination between the unknownprotein and the molecular weight markers. In one embodiment, thepolypeptide and fragmented peptide molecular weight markers of theinvention can be visualized using antibodies generated against thesemarkers and conventional immunoblotting techniques. This detection isperformed under conventional conditions that do not result in thedetection of the unknown protein. It is understood that it may not bepossible to generate antibodies against all polypeptide fragments of theinvention, since small peptides may not contain immunogenic epitopes. Itis further understood that not all antibodies will work in this assay;however, those antibodies which are able to bind polypeptides andfragments of the invention can be readily determined using conventionaltechniques.

[0223] The unknown protein is also visualized by using a conventionalstaining procedure. The molar excess of unknown protein to polypeptideor fragmented peptide molecular weight markers of the invention is suchthat the conventional staining procedure predominantly detects theunknown protein. The level of these polypeptide or fragmented peptidemolecular weight markers is such as to allow little or no detection ofthese markers by the conventional staining method. The preferred molarexcess of unknown protein to polypeptide molecular weight markers of theinvention is between 2 and 100,000 fold. More preferably, the preferredmolar excess of unknown protein to these polypeptide molecular weightmarkers is between 10 and 10,000 fold and especially between 100 and1,000 fold.

[0224] It is understood of course that many techniques can be used forthe determination and detection of molecular weight and isoelectricpoint of an unknown protein, polypeptides, and fragmented peptidesthereof using these polypeptide molecular weight markers and peptidefragments thereof and that these embodiments in no way limit the scopeof the invention.

[0225] In another embodiment, the analysis of the progressivefragmentation of the polypeptides of the invention into specificpeptides (D. W. Cleveland et al., J. Biol. Chem. 252:1102-1106, 1977),such as by altering the time or temperature of the fragmentationreaction, can be used as a control for the extent of cleavage of anunknown protein. For example, cleavage of the same amount of polypeptideand unknown protein under identical conditions can allow for a directcomparison of the extent of fragmentation. Conditions that result in thecomplete fragmentation of the polypeptide can also result in completefragmentation of the unknown protein.

[0226] As to the specific use of the polypeptides and fragmentedpeptides of the invention as molecular weight markers, the fragmentationof the IL-1 zeta polypeptide of SEQ ID NO:3 with cyanogen bromidegenerates a unique set of fragmented peptide molecular weight markerswith molecular weights of approximately 701.7, 2955.4, 5101.8 and12688.5 Daltons. Additionally, fragmentation of the Xrec2 polypeptide ofSEQ ID NO:4 with cyanogen bromide generates the following fragmentedpeptide molecular weight markers with molecular weights of approximately2216.7, 2259.6, 2376.6, 2738.1, 2901.1, 3417.2, 3627.1, 3656.1, 4042.5,4144.6, 4668.1, 4710.5, 4916.8, 5288.1, 6089.5, 8199.1, and 11919.7Daltons in the absence of glycosylation. In the fragmentation of bothSEQ ID NOs:3 and 4, an additional fragment of 149.2 Daltons results ifthe initiating methionine is present. The distribution of methionineresidues determines the number of amino acids in each peptide and theunique amino acid composition of each peptide determines its molecularweight.

[0227] In addition, the preferred purified polypeptides of the invention(SEQ ID NOs:3 and 4) have a calculated molecular weight of approximately21542.56 and 79967.85 Daltons, respectively. Thus, where an intactprotein is used, the use of these polypeptide molecular weight markersallows increased accuracy in the determination of apparent molecularweight of proteins that have apparent molecular weights close to21542.56 and 79967.85 Daltons. Where fragments are used, there isincreased accuracy in determining molecular weight over the range of themolecular weights of the fragment.

[0228] Finally, as to the kits that are encompassed by the invention,the constituents of such kits can be varied, but typically contain thepolypeptide and fragmented peptide molecular weight markers. Also, suchkits can contain the polypeptides wherein a site necessary forfragmentation has been removed. Furthermore, the kits can containreagents for the specific cleavage of the polypeptide and the unknownprotein by chemical or enzymatic cleavage. Kits can further containantibodies directed against polypeptides or fragments thereof of theinvention.

[0229] Identification of Unknown Proteins

[0230] As set forth above, a polypeptide or peptide fingerprint can beentered into or compared to a database of known proteins to assist inthe identification of the unknown protein using mass spectrometry (W. J.Henzel et al., Proc. Natl. Acad. Sci. USA 90:5011-5015, 1993; D. Fenyoet al., Electrophoresis 19:998-1005, 1998). A variety of computersoftware programs to facilitate these comparisons are accessible via theInternet, such as Protein Prospector (Internet site:prospector.uscf.edu), MultiIdent (Internet site:www.expasy.ch/sprot/multiident.html), PeptideSearch (Internetsite:www.mann.embl-heiedelberg.de...deSearch/FR_PeptideSearchForm.html), and ProFound (Internetsite:www.chait-sgi.rockefeller.edu/cgi-bin/prot-id-frag.html). Theseprograms allow the user to specify the cleavage agent and the molecularweights of the fragmented peptides within a designated tolerance. Theprograms compare observed molecular weights to predicted peptidemolecular weights derived from sequence databases to assist indetermining the identity of the unknown protein.

[0231] In addition, a polypeptide or peptide digest can be sequencedusing tandem mass spectrometry (MS/MS) and the resulting sequencesearched against databases (J. K. Eng, et al., J. Am. Soc. Mass Spec.5:976-989 (1994); M. Mann and M. Wilm, Anal. Chem. 66:4390-4399 (1994);J. A. Taylor and R. S. Johnson, Rapid Comm. Mass Spec. 11:1067-1075(1997)). Searching programs that can be used in this process exist onthe Internet, such as Lutefisk 97 (Internet site:www.lsbc.com:70/Lutefisk97.html), and the Protein Prospector, PeptideSearch and ProFound programs described above.

[0232] Therefore, adding the sequence of a gene and its predictedprotein sequence and peptide fragments to a sequence database can aid inthe identification of unknown proteins using mass spectrometry.

[0233] Antibodies

[0234] Antibodies that are immunoreactive with the polypeptides of theinvention are provided herein. Such antibodies specifically bind to thepolypeptides via the antigen-binding sites of the antibody (as opposedto non-specific binding). Thus, the polypeptides, fragments, variants,fusion proteins, etc., as set forth above may be employed as“immunogens” in producing antibodies immunoreactive therewith. Morespecifically, the polypeptides, fragment, variants, fusion proteins,etc. contain antigenic determinants or epitopes that elicit theformation of antibodies.

[0235] These antigenic determinants or epitopes can be either linear orconformational (discontinuous). Linear epitopes are composed of a singlesection of amino acids of the polypeptide, while conformational ordiscontinuous epitopes are composed of amino acids sections fromdifferent regions of the polypeptide chain that are brought into closeproximity upon protein folding (C. A. Janeway, Jr. and P. Travers,Immuno Biology 3:9 (Garland Publishing Inc., 2nd ed. 1996)). Becausefolded proteins have complex surfaces, the number of epitopes availableis quite numerous; however, due to the conformation of the protein andsteric hindrances, the number of antibodies that actually bind to theepitopes is less than the number of available epitopes (C. A. Janeway,Jr. and P. Travers, Immuno Biology 2:14 (Garland Publishing Inc., 2nded. 1996)). Epitopes may be identified by any of the methods known inthe art.

[0236] Thus, one aspect of the present invention relates to theantigenic epitopes of the polypeptides of the invention. Such epitopesare useful for raising antibodies, in particular monoclonal antibodies,as described in more detail below. Additionally, epitopes from thepolypeptides of the invention can be used as research reagents, inassays, and to purify specific binding antibodies from substances suchas polyclonal sera or supernatants from cultured hybridomas. Suchepitopes or variants thereof can be produced using techniques well knownin the art such as solid-phase synthesis, chemical or enzymatic cleavageof a polypeptide, or using recombinant DNA technology.

[0237] As to the antibodies that can be elicited by the epitopes of thepolypeptides of the invention, whether the epitopes have been isolatedor remain part of the polypeptides, both polyclonal and monoclonalantibodies may be prepared by conventional techniques. See, for example,Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Kennet et al. (eds.), Plenum Press, New York (1980); andAntibodies: A Laboratory Manual, Harlow and Land (eds.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).

[0238] Hybridoma cell lines that produce monoclonal antibodies specificfor the polypeptides of the invention are also contemplated herein. Suchhybridomas may be produced and identified by conventional techniques.One method for producing such a hybridoma cell line comprises immunizingan animal with a polypeptide; harvesting spleen cells from the immunizedanimal; fusing said spleen cells to a myeloma cell line, therebygenerating hybridoma cells; and identifying a hybridoma cell line thatproduces a monoclonal antibody that binds the polypeptide. Themonoclonal antibodies may be recovered by conventional techniques.

[0239] The monoclonal antibodies of the present invention includechimeric antibodies, e.g., humanized versions of murine monoclonalantibodies. Such humanized antibodies may be prepared by knowntechniques and offer the advantage of reduced immunogenicity when theantibodies are administered to humans. In one embodiment, a humanizedmonoclonal antibody comprises the variable region of a murine antibody(or just the antigen binding site thereof) and a constant region derivedfrom a human antibody. Alternatively, a humanized antibody fragment maycomprise the antigen binding site of a murine monoclonal antibody and avariable region fragment (lacking the antigen-binding site) derived froma human antibody. Procedures for the production of chimeric and furtherengineered monoclonal antibodies include those described in Riechmann etal. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick etal. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS 14:139,May, 1993). Procedures to generate antibodies transgenically can befound in GB 2,272,440, U.S. Pat. Nos. 5,569,825 and 5,545,806 andrelated patents claiming priority therefrom, all of which areincorporated by reference herein.

[0240] Antigen-binding fragments of the antibodies, which may beproduced by conventional techniques, are also encompassed by the presentinvention. Examples of such fragments include, but are not limited to,Fab and F(ab′)₂ fragments. Antibody fragments and derivatives producedby genetic engineering techniques are also provided.

[0241] In one embodiment, the antibodies are specific for thepolypeptides of the present invention and do not cross-react with otherproteins. Screening procedures by which such antibodies may beidentified are well known, and may involve immunoaffinitychromatography, for example.

[0242] Uses Thereof

[0243] The antibodies of the invention can be used in assays to detectthe presence of the polypeptides or fragments of the invention, eitherin vitro or in vivo. The antibodies also may be employed in purifyingpolypeptides or fragments of the invention by immunoaffinitychromatography. Those antibodies that additionally can block binding ofthe polypeptides of the invention to the binding partner may be used toinhibit a biological activity that results from such binding. Suchblocking antibodies may be identified using any suitable assayprocedure, such as by testing antibodies for the ability to inhibitbinding of IL-1 zeta to certain cells expressing the IL-1 zetareceptors. Alternatively, blocking antibodies may be identified inassays for the ability to inhibit a biological effect that results frompolypeptides of the invention binding to their binding partners totarget cells. Antibodies may be assayed for the ability to inhibit IL-1zeta-mediated, Xrec2-mediated, or binding partner-mediated cell lysis,for example. Antibodies that are antagonistic or block IL-1 zetaactivity are useful as therapeutic agents for downregulating IL-12expression and TNF expression. Thus, such antagonists are useful intreating deleterious affects of inflammation and disease associated withadverse immune responses as described herein. Similarly, agonisticantibodies to IL-1 zeta polypeptide are useful in upregulating IL-12expression and are useful in enhancing the effects of Th1 mediatedimmune response as described herein.

[0244] Such an antibody may be employed in an in vitro procedure, oradministered in vivo to inhibit a biological activity mediated by theentity that generated the antibody. Disorders caused or exacerbated(directly or indirectly) by the interaction of the polypeptides of theinvention with the binding partner thus may be treated. A therapeuticmethod involves in vivo administration of a blocking antibody to amammal in an amount effective in inhibiting a binding partner-mediatedbiological activity or a biological activity such as the inhibition ofIL-12 and TNF expression. Monoclonal antibodies are generally preferredfor use in such therapeutic methods. In one embodiment, anantigen-binding antibody fragment is employed.

[0245] Antibodies may be screened for agonistic (i.e., ligand-mimicking)properties. Such antibodies, upon binding to cell surface receptor,induce biological effects (e.g., transduction of biological signals)similar to the biological effects induced when IL-1 binds to cellsurface IL-1 receptors. Agonistic antibodies may be used to activateIL-12 expression and treat disease associated with Th1 mediatedpathways.

[0246] Compositions comprising an antibody that is directed againstpolypeptides of the invention, and a physiologically acceptable diluent,excipient, or carrier, are provided herein. Suitable components of suchcompositions are as described above for compositions containingpolypeptides of the invention.

[0247] Also provided herein are conjugates comprising a detectable(e.g., diagnostic) or therapeutic agent, attached to the antibody.Examples of such agents are presented above. The conjugates find use inin vitro or in vivo procedures.

[0248] Because the IL-1 zeta polypeptides, and particularly the TDZ1isoform, are active in IL-12 regulation and TNF regulation, inhibitorssuch as small molecule inhibitors of its function or its proteinassociations (or antisense or other inhibitors of its synthesis) will beuseful in treating autoimmune and/or inflammatory disorders.Accordingly, IL-1 zeta polypeptides and fragments of IL-1 zetapolypeptides that are capable of upregulating IL-12 production or TNFproduction as described below, for example, are useful in screeningassays to identify compounds and small molecules which inhibit(antagonize) functions and activities of IL-1 zeta polypeptide anddescribed herein. Similarly, IL-1 zeta polypeptides and fragments ofIL-1 zeta polypeptides that are capable of upregulating IL-12 productionare useful in screening assays to identify compounds and small moleculeswhich agonize or enhance IL-12 expression. Such compounds are useful astherapeutics for the herein described uses associated with enhancedIL-12 expression. (U.S. Pat. No. 5,674,483 and U.S. Pat. No. 5,928,636which are incorporated herein by reference).

[0249] Thus, for example, polypeptides and polypeptide fragments of theinvention may be used to identify antagonists and agonists from cells,cell-free preparations, chemical libraries, and natural productmixtures. The antagonists and agonists may be natural or modifiedsubstrates, ligands, enzymes, receptors, etc. of the polypeptides of theinstant invention, or may be structural or functional mimetics of thepolypeptides. Potential antagonists of the instant invention may includesmall molecules, peptides and antibodies that bind to and occupy abinding site of the inventive polypeptides or a binding partner thereof,causing them to be unavailable to bind to their natural binding partnersand therefore preventing normal biological activity. Potential agonistsinclude small molecules, peptides and antibodies which bind to theinstant polypeptides or binding partners thereof, and elicit the same orenhanced biologic effects as those caused by the binding of thepolypeptides of the instant invention.

[0250] Small molecule agonists and antagonists are usually less than 10Kmolecular weight and may possess a number of physicochemical andpharmacological properties which enhance cell penetration, resistdegradation and prolong their physiological half-lives (Gibbs, J.,Pharmaceutical Research in Molecular Oncology, Cell, Vol. 79 (1994)).Antibodies, which include intact molecules as well as fragments such asFab and F(ab′)2 fragments, as well as recombinant molecules derivedtherefrom, may be used to bind to and inhibit the polypeptides of theinstant invention by blocking the propagation of a signaling cascade. Itis preferable that the antibodies are humanized, and more preferablethat the antibodies are human. The antibodies of the present inventionmay be prepared by any of a variety of well-known methods.

[0251] Screening methods are known in the art and along with integratedrobotic systems and collections of chemical compounds/natural productsare extensively incorporated in high throughput screening so that largenumbers of test compounds can be tested for antagonist or agonistactivity within a short amount of time. These methods includehomogeneous assay formats such as fluorescence resonance energytransfer, fluorescence polarization, time-resolved fluorescenceresonance energy transfer, scintillation proximity assays, reporter geneassays, fluorescence quenched enzyme substrate, chromogenic enzymesubstrate and electrochemiluminescence, as well as more traditionalheterogeneous assay formats such as enzyme-linked immunosorbant assays(ELISA) or radioimmunoassays.

[0252] Homogeneous assays are mix-and-read style assays that are veryamenable to robotic application, whereas heterogeneous assays requireseparation of free from bound analyte by more complex unit operationssuch as filtration, centrifugation or washing. These assays are utilizedto detect a wide variety of specific biomolecular interactions and theinhibition thereof by small organic molecules, includingprotein-protein, receptor-ligand, enzyme-substrate, and so on. Theseassay methods and techniques are well known in the art (see, e.g., HighThroughput Screening: The Discovery of Bioactive Substances, John P.Devlin (ed.), Marcel Dekker, New York, 1997 ISBN: 0-8247-0067-8). Thescreening assays of the present invention are amenable to highthroughput screening of chemical libraries and are suitable forscreening test compounds in order to identify small molecule drugcandidates, antibodies, peptides, and other antagonists and/or agonists,natural or synthetic.

[0253] Thus, a method of the present invention includes screening a testcompound to determine its effect on the ability of a polypeptide of thisinvention to increase or decrease IL-12 expression and/or TNFexpression. Such a method involves co-culturing an IL-1 zeta polypeptideof this invention, particularly the TDZ1 isoform, and cells capable ofexpressing IL-12 and/or TNF (e.g. monocytes, PBMC) and analyzing theculture for IL-12 and/or TNF levels. If the level of expression differsfrom that level of expression that is observed in the absence of testcompound, a test compound that affects IL-12 and/or TNF expression isidentified. Polypeptides that are useful in the screening methodsinclude the IL-1 zeta polypeptides of this invention and fragments ofthe IL-1 zeta polypeptides that upregulate IL-12 expression and/or TNFexpression, particularly the TDZ1 isoform.

[0254] In one embodiment of a method for identifying molecules whichinhibit or antagonize the polypeptides of this invention involves addinga test compound to a medium which contains cells that express thepolypeptides of the instant invention; changing the conditions of themedium so that, but for the presence of the test compound, thepolypeptides would be bound to their natural ligands, substrates oreffector molecules, and observing the binding and stimulation orinhibition of a functional response. The activity of the cells whichwere contacted with the test compound may then be compared with theidentical cells which were not contacted and antagonists and agonists ofthe polypeptides of the instant invention may be identified. Themeasurement of biological activity may be performed by a number ofwell-known methods such as measuring the amount of protein present (e.g.an ELISA) or measuring the protein's activity. A decrease in biologicalstimulation or activation indicates an antagonist. An increase indicatesan agonist.

[0255] Another embodiment of the invention relates to uses ofpolypeptides of this invention to study cell signal transduction.Cellular signaling often involves a molecular activation cascade, duringwhich a receptor propagates a ligand-receptor mediated signal byspecifically activating intracellular kinases which phosphorylate targetsubstrates. These substrates can themselves be kinases which becomeactivated following phosphorylation. Alternatively, they can be adaptermolecules that facilitate down stream signaling through protein-proteininteraction following phosphorylation. Accordingly, these polypeptidesand active fragments can be used as reagents to identify novel moleculesinvolved in signal transduction pathways.

[0256] As therapeutics, inhibitors or agonists of IL-1 zeta activity canbe administered to agonize or antagonize IL-1 zeta activity, thusproviding useful immunoregulators. Various liposome-based compositionsof the inventive polypeptides are envisioned herein.

[0257] Inhibitors and enhancers of the polypeptides or polypeptidefragments having biological activity are useful in treating a variety ofmedical conditions. IL-1 zeta polypeptides are associated with IL-12production and dysregulation of IL-12 production, and thus agonists ofIL-1 zeta polypeptides are useful for treating diseases and medicalconditions that are therapeutically responsive to IL-12 expression. Suchdiseases and medical conditions include infectious diseases, such asHIV, Hepatitis B and Hepatitis C, papilloma, etc.; and, bacterialinfections, including tuberculosis, salmonellosis, listeriousis; and,parasitic infections such as malaria, leishmaniasis and schistosomiasis.Agonists are also useful for treating dysregulated immune response, e.g.use as a vaccine (e.g. for use in connection with antigen such as formeasles vaccination) or vaccine adjuvant, increased response tobacterial and viral infection, as just discussed, and as therapeuticimmunotherapies including anticancer immunotherapy treatments. (See U.S.Pat. Nos. 6,086,876, and 6,168,923 both of which are incorporated hereinby reference) In another embodiment, agonists of IL-1 zeta polypeptidescan be administered in combination with other agents or cytokines fortreating disease and medical conditions. For example, agonists can beadministered in combination with IFN or IFN alpha. Antagonists of IL-1zeta polypeptides are useful in treating certain types of immune systemdysfunction associated with IL-12 dysregulation such as autoimmunediseases, inflammatory conditions, complications that are associatedwith bacterial infections that occur with increased IL-12 activity andconditions associated with increased expression or activity of IL-12.Thus, therapeutics discovered by screening IL-1 zeta polypeptides, theTDZ1 isoform and active fragments for agonistic or antagonistic activityhave properties that make them suitable for use as: anti-inflammatory,anti-tumor or anti-cancer, anti-bacterial, and anti-viral.

[0258] Compositions of the present invention may contain a polypeptideor and antagonist or agonist in any form described herein, such asnative proteins, variants, derivatives, oligomers, biologically activefragments of the compounds described herein, small molecules,antibodies, etc. In particular embodiments, the composition comprisespeptides, small molecules, antibodies or oligomers comprising solublepolypeptides.

[0259] Compositions comprising an effective amount of a polypeptide ofthe present invention, in combination with other components such as aphysiologically acceptable diluent, carrier, or excipient, are providedherein. The polypeptides can be formulated according to known methodsused to prepare pharmaceutically useful compositions. They can becombined in admixture, either as the sole active material or with otherknown active materials suitable for a given indication, withpharmaceutically acceptable diluents (e.g., saline, Tris-HCl, acetate,and phosphate buffered solutions), preservatives (e.g., thimerosal,benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/orcarriers. Suitable formulations for pharmaceutical compositions includethose described in Remington's Pharmaceutical Sciences, 16th ed. 1980,Mack Publishing Company, Easton, Pa.

[0260] In addition, such compositions can be complexed with polyethyleneglycol (PEG), metal ions, or incorporated into polymeric compounds suchas polyacetic acid, polyglycolic acid, hydrogels, dextran, etc., orincorporated into liposomes, microemulsions, micelles, unilamellar ormultilamellar vesicles, erythrocyte ghosts or spheroblasts. Suchcompositions will influence the physical state, solubility, stability,rate of in vivo release, and rate of in vivo clearance, and are thuschosen according to the intended application.

[0261] The compositions of the invention can be administered in anysuitable manner, e.g., topically, parenterally, orally, intracraniallyor by inhalation. The term “parenteral” includes injection, e.g., bysubcutaneous, intravenous, or intramuscular routes, also includinglocalized administration, e.g., at a site of disease or injury (forexample, intracoronary or intra tumor administration or injection into ajoint undergoing an inflammatory reaction). Sustained release fromimplants is also contemplated. One skilled in the pertinent art willrecognize that suitable dosages will vary, depending upon such factorsas the nature of the disorder to be treated, the patient's body weight,age, and general condition, and the route of administration. Preliminarydoses can be determined according to animal tests, and the scaling ofdosages for human administration is performed according to art-acceptedpractices.

[0262] Moreover, it has been found that DNA encoding a polypeptide canbe administered to a mammal in such a way that it is taken up by cells,and expressed. The resultant protein will then be available to exert atherapeutic effect. Accordingly, compositions comprising nucleic acidsin physiologically acceptable formulations are also contemplated. DNAmay be formulated for injection, for example.

[0263] The following examples are provided to further illustrateparticular embodiments of the invention, and are not to be construed aslimiting the scope of the present invention.

EXAMPLE 1 Isolation of the IL-1 Zeta and Xrec2 Nucleic Acids

[0264] Human IL-1 zeta nucleic acid sequence was obtained by sequencingEST IMAGE clone 1628761, accession #AI014548, which encoded a partialopen reading frame (ORF). A number of cDNA libraries were screened withinternal primers to determine the expression pattern of the polypeptide.After performing PCR using two internal primers of human IL-1 zetasequence, the following cDNA libraries were positive for IL-1 zetasequences: bone marrow stromal, human pancreatic tumor, and Raji. IL-1zeta clones were isolated from human genomic DNA sequences, bone marrowstromal and human pancreatic tumor libraries, and sequenced.

[0265] Human Xrec2 sequences were obtained by high-throughputsequencing, PCR, and 5′RACE reactions. High-throughput shotgunsequencing of chromosome region Xp11 yielded sequences for exons 4-6 ofXrec2 (Genbank accession numbers AL031466 and AL031575). Similarly,sequence of chromosome region Xp22-164-166 (Genbank accession numberAC005748) yielded sequences for exons 10-12 of Xrec2.

[0266] PCR performed on human brain first strand cDNA using primerswithin exons 5 and 11 generated sequence for exons 7-9. 5′RACE reactionswere then performed using testis cDNA and nested primers within exon 4to obtain exon 3 sequences which contained the predicted initiatormethionine. Both PCR and the 5′RACE reactions were performed usingstandard protocols.

EXAMPLE 2 Use of Purified IL-1 Zeta and Xrec2 Polypeptides

[0267] Polypeptide-Specific ELISA:

[0268] Serial dilutions of IL-1 zeta- or Xrec2-containing samples (in 50mM NaHCO₃, brought to pH 9 with NaOH) are coated onto Linbro/Titertek 96well flat bottom E.I.A. microtitration plates (ICN Biomedicals Inc.,Aurora, Ohio) at 100:1/well. After incubation at 4EC for 16 hours, thewells are washed six times with 200:1 PBS containing 0.05% Tween-20(PBS-Tween). The wells are then incubated with FLAG7-binding partner at1 mg/ml in PBS-Tween with 5% fetal calf serum (FCS) for 90 minutes(100:1 per well), followed by washing as above. Next, each well isincubated with the anti-FLAG7 (monoclonal antibody M2 at 1 mg/ml inPBS-Tween containing 5% FCS for 90 minutes (100:1 per well), followed bywashing as above. Subsequently, wells are incubated with a polyclonalgoat anti-mIgG1-specific horseradish peroxidase-conjugated antibody (a1:5000 dilution of the commercial stock in PBS-Tween containing 5% FCS)for 90 minutes (100:1 per well). The HRP-conjugated antibody is obtainedfrom Southern Biotechnology Associates, Inc., Birmingham, Ala. Wellsthen are washed six times, as above.

[0269] For development of the ELISA, a substrate mix [100:1 per well ofa 1:1 premix of the TMB Peroxidase Substrate and Peroxidase Solution B(Kirkegaard Perry Laboratories, Gaithersburg, Md.)] is added to thewells. After sufficient color reaction, the enzymatic reaction isterminated by addition of 2 N H₂SO₄ (50:1 per well). Color intensity(indicating ligand receptor binding) is determined by measuringextinction at 450 nm on a V Max plate reader (Molecular Devices,Sunnyvale, Calif.).

EXAMPLE 3 Amino Acid Sequence

[0270] The amino acid sequence of IL-1 zeta and Xrec2 were determined bytranslation of the complete nucleotide sequences of SEQ ID NOs:1 and 2,respectively.

EXAMPLE 4 DNA and Amino Acid Sequences

[0271] The IL-1 zeta and Xrec2 nucleic acid sequences were determined bystandard double stranded sequencing of the composite sequence of ESTIMAGE clones (accession #AI014548 (IL-1 zeta) and #AL031575 and#AC005748 (Xrec2)), and of additional sequences obtained from PCR and5′RACE reactions.

[0272] The nucleotide sequence of the isolated IL-1 zeta and Xrec2 DNAand the amino acid sequence encoded thereby, are presented in SEQ IDNOs: 1-4. The sequence of the IL-1 zeta DNA fragment isolated by PCRcorresponds to nucleotides 1 to 579 of SEQ ID NO:1, which encode aminoacids 1 to 192 of SEQ ID NO:3; and the sequence of the Xrec2 DNAfragment also isolated by PCR corresponds to nucleotides 1 to 2088 ofSEQ ID NO:2, which encode amino acids 1 to 698 of SEQ ID NO:4.

[0273] The amino acid sequences of SEQ ID NOs:3 and 4 bear significanthomology to other known IL-1 ligand and receptor family members,respectively.

EXAMPLE 5 Monoclonal Antibodies that Bind Polypeptides of the Invention

[0274] This example illustrates a method for preparing monoclonalantibodies that bind IL-1 zeta. The same protocol can be used to producemonoclonal antibodies that bind Xrec2. Suitable immunogens that may beemployed in generating such antibodies include, but are not limited to,purified IL-1 zeta polypeptide or an immunogenic fragment thereof suchas the extracellular domain, or fusion proteins containing IL-1 zeta(e.g., a soluble IL-1 zeta/Fc fusion protein).

[0275] Purified IL-1 zeta can be used to generate monoclonal antibodiesimmunoreactive therewith, using conventional techniques such as thosedescribed in U.S. Pat. No. 4,411,993. Briefly, mice are immunized withIL-1 zeta immunogen emulsified in complete Freund's adjuvant, andinjected in amounts ranging from 10-100 g subcutaneously orintraperitoneally. Ten to twelve days later, the immunized animals areboosted with additional IL-1 zeta emulsified in incomplete Freund'sadjuvant. Mice are periodically boosted thereafter on a weekly tobi-weekly immunization schedule. Serum samples are periodically taken byretro-orbital bleeding or tail-tip excision to test for IL-1 zetaantibodies by dot blot assay, ELISA (Enzyme-Linked Immunosorbent Assay)or inhibition of IL-1 zeta receptor binding.

[0276] Following detection of an appropriate antibody titer, positiveanimals are provided one last intravenous injection of IL-1 zeta insaline. Three to four days later, the animals are sacrificed, spleencells harvested, and spleen cells are fused to a murine myeloma cellline, e.g., NS1 or preferably P3x63Ag8.653 (ATCC CRL 1580). Fusionsgenerate hybridoma cells, which are plated in multiple microtiter platesin a HAT (hypoxanthine, aminopterin and thymidine) selective medium toinhibit proliferation of non-fused cells, myeloma hybrids, and spleencell hybrids.

[0277] The hybridoma cells are screened by ELISA for reactivity againstpurified IL-1 zeta by adaptations of the techniques disclosed in Engvallet al., (Immunochem. 8:871, 1971) and in U.S. Pat. No. 4,703,004. Apreferred screening technique is the antibody capture techniquedescribed in Beckmann et al., (J. Immunol. 144:4212, 1990). Positivehybridoma cells can be injected intraperitoneally into syngeneic BALB/cmice to produce ascites containing high concentrations of anti-IL-1 zetamonoclonal antibodies. Alternatively, hybridoma cells can be grown invitro in flasks or roller bottles by various techniques. Monoclonalantibodies produced in mouse ascites can be purified by ammonium sulfateprecipitation, followed by gel exclusion chromatography. Alternatively,affinity chromatography based upon binding of antibody to Protein A orProtein G can also be used, as can affinity chromatography based uponbinding to IL-1 zeta.

EXAMPLE 6 Northern Blot Analysis

[0278] The tissue distribution of IL-1 zeta and Xrec2 mRNA isinvestigated by Northern blot analysis, as follows. An aliquot of aradiolabeled probe is added to two different human multiple tissueNorthern blots (Clontech, Palo Alto, Calif.; Biochain, Palo Alto,Calif.). The blots are hybridized in 10× Denhardts, 50 mM Tris pH 7.5,900 mM NaCl, 0.1% Na pyrophosphate, 1% SDS, 200 g/mL salmon sperm DNA.Hybridization is conducted overnight at 63EC in 50% formamide aspreviously described (March et al., Nature 315:641-647, 1985). The blotsare then washed with 2×SSC, 0.1% SDS at 68EC for 30 minutes. The cellsand tissues with the highest levels of IL-1 zeta and Xrec2 mRNA aredetermined by comparison to control probing with a β-actin-specificprobe.

EXAMPLE 7 Binding Assay

[0279] Full length IL-1 zeta can be expressed and tested for the abilityto bind IL-1 zeta receptors. The binding assay can be conducted asfollows.

[0280] A fusion protein comprising a leucine zipper peptide fused to theN-terminus of a soluble IL-1 zeta polypeptide (LZ-IL-1 zeta) is employedin the assay. An expression construct is prepared, essentially asdescribed for preparation of the FLAG⁷(IL-1 zeta) expression constructin Wiley et al. (Immunity, 3:673-682, 1995; hereby incorporated byreference), except that DNA encoding the FLAG⁷ peptide was replaced witha sequence encoding a modified leucine zipper that allows fortrimerization. The construct, in expression vector pDC409, encodes aleader sequence derived from human cytomegalovirus, followed by theleucine zipper moiety fused to the N-terminus of a soluble IL-1 zetapolypeptide. The LZ-IL-1 zeta is expressed in CHO cells, and purifiedfrom the culture supernatant.

[0281] The expression vector designated pDC409 is a mammalian expressionvector derived from the pDC406 vector described in McMahan et al. (EMBOJ. 10:2821-2832, 1991; hereby incorporated by reference). Features addedto pDC409 (compared to pDC406) include additional unique restrictionsites in the multiple cloning site (mcs); three stop codons (one in eachreading frame) positioned downstream of the mcs; and a T7 polymerasepromoter, downstream of the mcs, that facilitates sequencing of DNAinserted into the mcs.

[0282] For expression of full length human IL-1 zeta protein, the entirecoding region (i.e., the DNA sequence presented in SEQ ID NO:1) isamplified by polymerase chain reaction (PCR). The template employed inthe PCR is the cDNA clone isolated from a (pancreatic tumor) cDNAlibrary, as described in example 1. The isolated and amplified DNA isinserted into the expression vector pDC409, to yield a constructdesignated pDC409-IL-1 zeta.

[0283] LZ-IL-1 zeta polypeptide is employed to test the ability to bindto host cells expressing recombinant or endogenous IL-1 zeta receptors,as discussed above. Cells expressing IL-1 zeta receptor are cultured inDMEM supplemented with 10% fetal bovine serum, penicillin, streptomycin,and glutamine. Cells are incubated with LZ-IL-1 zeta (5 mg/ml) for about1 hour. Following incubation, the cells are washed to remove unboundLZ-IL-1 zeta and incubated with a biotinylated anti-LZ monoclonalantibody (5 mg/ml), and phycoerythrin-conjugated streptavidin (1:400),before analysis by fluorescence-activated cell scanning (FACS). Thecytometric analysis was conducted on a FACscan (Beckton Dickinson, SanJose, Calif.).

[0284] The cells expressing IL-1 zeta receptors showed significantlyenhanced binding of LZ-IL-1 zeta, compared to the control cells notexpressing IL-1 zeta receptors.

EXAMPLE 8 Obtaining TDZ.1, TDZ.2, and TDZ.3 and Tissue Distribution

[0285] In order to determine and study the relative abundance and tissuedistribution of Tango-77 (WO 99/06426), an alternatively spliced form ofIL-1 zeta, and IL-1 zeta, RT-PCR was performed. The primers used in theRT PCR were 5′ primers specific for either Tango-77 exon #1 (see FIG. 1)or IL-1 zeta exon #1 (exons #3 in FIG. 1) in combination with a common3′ primer from the common final exon (exon #6 in FIG. 1). The PCRreactions were performed using first strand cDNA from multiple humantissue sources purchased from Clontech, Palo Alto, Calif. The PCRreaction generated PCR products that included the predicted size productand additional bands. In particular, three different sized PCR productswere isolated and used to obtain sequence information from multipletissue cDNAs. The sequences of these three products, SEQ ID NOs:5, 6, 7and encoded amino acids of SEQ ID NO:8, 9, and 10, are splice variants.The organization the relationship of these splice variants are shown inFIG. 1 and discussed above. The splice variants are TDZ.1, TDZ.2, andTDZ.3 (Testis-Derived Zeta variants) because all three of them areexpressed in testis. Testis is a common expression tissue. However, itis not the only expression tissue. Table II describes the results of thetissue expression study for Tango-77, IL-1 zeta, TDZ.1, TDZ.2, andTDZ.3. TDZ.1 and TDZ.2 contain exons 4, 5 and 6 which correspond to thelast three exons of IL-1 zeta and correspond to the conserved structuraldomain of the molecule. As discussed above, when aligned with othermembers of the IL-1 family, exons 4, 5 and 6 are shown to contain manyconserved residues within conserved structural motifs.

[0286] A polymorphism of Tango 77 in exon #2 of FIG. 1 is noted. In theisolated cDNAS a valine occurs in lieu of a glycine at the third residueof exon #2. In the Tango-77 sequence, the amino acid sequence isPAGSPLEP. In the polymorphism the sequence is PAVSPLEP.

[0287] Tissue Distribution of FIL-1Z Splice Variants TABLE II TissueIL-1z Tango-77 TDZ.1 TDZ.2 TDZ.3 kidney − − + − − pancreas − − − − −skeletal muscle − − + − − heart − + − − − testis + + + + + prostrate +− + − − spleen − − − − − ovary − + + − − thymus − − − − − colon + + + −− leukocytes − − − − − small intestine − + + − − liver − + + − − brain +− − − − placenta + + + − + lung + + + − + tonsil − + + − − fetalliver + + + − − lymph node + + + − − bone marrow − + + + +

EXAMPLE 9 IL-1 Zeta Polypeptide Induces TNF and IL-12 Secretion

[0288] The following assays were performed to study cytokine inductionby IL-1 Zeta polypeptides. A protein of IL-1 zeta, TDZ.1 isoform, fusedto a FLAG-poly His polypeptide at its C-terminus, was prepared andco-cultured with human monocytes. Varying concentrations of the TDZ.1isoform were used with a lower level concentration of 5 nM. The culturewas analyzed for cytokines and found to have increased levels ofTNF-alpha and IL-12. This cytokine inducing activity was dose dependent.

[0289] The references cited herein are incorporated by reference hereinin their entirety.

1 15 1 579 DNA Homo sapiens 1 atgtcaggct gtgataggag ggaaacagaaaccaaaggaa agaacagctt taagaagcgc 60 ttaagaggtc caaaggtgaa gaacttaaacccgaagaaat tcagcattca tgaccaggat 120 cacaaagtac tggtcctgga ctctgggaatctcatagcag ttccagataa aaactacata 180 cgcccagaga tcttctttgc attagcctcatccttgagct cagcctctgc ggagaaagga 240 agtccgattc tcctgggggt ctctaaaggggagttttgtc tctactgtga caaggataaa 300 ggacaaagtc atccatccct tcagctgaagaaggagaaac tgatgaagct ggctgcccaa 360 aaggaatcag cacgccggcc cttcatcttttatagggctc aggtgggctc ctggaacatg 420 ctggagtcgg cggctcaccc cggatggttcatctgcacct cctgcaattg taatgagcct 480 gttggggtga cagataaatt tgagaacaggaaacacattg aattttcatt tcaaccagtt 540 tgcaaagctg aaatgagccc cagtgaggtcagcgattag 579 2 2091 DNA Homo sapiens 2 atgaaagctc cgattccaca cttgattctcttatacgcta cttttactca gagtttgaag 60 gttgtgacca aaagaggctc cgccgatggatgcactgact ggtctatcga tatcaagaaa 120 tatcaagttt tggtgggaga gcctgttcgaatcaaatgtg cactctttta tggttatatc 180 agaacaaatt actcccttgc ccaaagtgctggactcagtt tgatgtggta caaaagttct 240 ggtcctggag actttgaaga gccaatagcctttgacggaa gtagaatgag caaagaagaa 300 gactccattt ggttccggcc aacattgctacaggacagtg gtctctacgc ctgtgtcatc 360 agaaactcca cttactgtat gaaagtatccatctcactga cagtgggtga aaatgacact 420 ggactctgct ataattccaa gatgaagtattttgaaaaag ctgaacttag caaaagcaag 480 gaaatttcat gccgtgacat agaggattttctactgccaa ccagagaacc tgaaatcctt 540 tggtacaagg aatgcaggac aaaaacatggaggccaagta ttgtattcaa aagagatact 600 ctgcttataa gagaagtcag agaagatgacattggaaatt atacctgtga attaaaatat 660 ggaggctttg ttgtgagaag aactactgaattaactgtta cagcccctct gactgataag 720 ccacccaagc ttttgtatcc tatggaaagtaaactgacaa ttcaggagac ccagctgggt 780 gactctgcta atctaacctg cagagctttctttgggtaca gcggagatgt cagtccttta 840 atttactgga tgaaaggaga aaaatttattgaagatctgg atgaaaatcg agtttgggaa 900 agtgacatta gaattcttaa ggagcatcttggggaacagg aagtttccat ctcattaatt 960 gtggactctg tggaagaagg tgacttgggaaattactcct gttatgttga aaatggaaat 1020 ggacgtcgac acgccagcgt tctccttcataaacgagagc taatgtacac agtggaactt 1080 gctggaggcc ttggtgctat actcttgctgcttgtatgtt tggtgaccat ctacaagtgt 1140 tacaagatag aaatcatgct cttctacaggaatcattttg gagctgaaga gctcgatgga 1200 gacaataaag attatgatgc atacttatcatacaccaaag tggatcctga ccagtggaat 1260 caagagactg gggaagaaga acgttttgcccttgaaatcc tacctgatat gcttgaaaag 1320 cattatggat ataagttgtt tataccagatagagatttaa tcccaactgg aacatacatt 1380 gaagatgtgg caagatgtgt agatcaaagcaagcggctga ttattgtcat gaccccaaat 1440 tacgtagtta gaaggggctg gagcatctttgagctggaaa ccagacttcg aaatatgctt 1500 gtgactggag aaattaaagt gattctaattgaatgcagtg aactgagagg aattatgaac 1560 taccaggagg tggaggccct gaagcacaccatcaagctcc tgacggtcat taaatggcat 1620 ggaccaaaat gcaacaagtt gaactccaagttctggaaac gtttacagta tgaaatgcct 1680 tttaagagga tagaacccat tacacatgagcaggctttag atgtcagtga gcaagggcct 1740 tttggggagc tgcagactgt ctcggccatttccatggccg cggccacctc cacagctcta 1800 gccactgccc atccagatct ccgttctacctttcacaaca cgtaccattc acaaatgcgt 1860 cagaaacact actaccgaag ctatgagtacgacgtacctc ctaccggcac cctgcctctt 1920 acctccatag gcaatcagca tacctactgtaacatcccta tgacactcat caacgggcag 1980 cggccacaga caaaatcgag cagggagcagaatccagatg aggcccacac aaacagtgcc 2040 atcctgccgc tgttgccaag ggagaccagtatatccagtg tgatatggtg a 2091 3 192 PRT Homo sapiens 3 Met Ser Gly CysAsp Arg Arg Glu Thr Glu Thr Lys Gly Lys Asn Ser 1 5 10 15 Phe Lys LysArg Leu Arg Gly Pro Lys Val Lys Asn Leu Asn Pro Lys 20 25 30 Lys Phe SerIle His Asp Gln Asp His Lys Val Leu Val Leu Asp Ser 35 40 45 Gly Asn LeuIle Ala Val Pro Asp Lys Asn Tyr Ile Arg Pro Glu Ile 50 55 60 Phe Phe AlaLeu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly 65 70 75 80 Ser ProIle Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys 85 90 95 Asp LysAsp Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu 100 105 110 LysLeu Met Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe 115 120 125Ile Phe Tyr Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser Ala 130 135140 Ala His Pro Gly Trp Phe Ile Cys Thr Ser Cys Asn Cys Asn Glu Pro 145150 155 160 Val Gly Val Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu PheSer 165 170 175 Phe Gln Pro Val Cys Lys Ala Glu Met Ser Pro Ser Glu ValSer Asp 180 185 190 4 696 PRT Homo sapiens 4 Met Lys Ala Pro Ile Pro HisLeu Ile Leu Leu Tyr Ala Thr Phe Thr 1 5 10 15 Gln Ser Leu Lys Val ValThr Lys Arg Gly Ser Ala Asp Gly Cys Thr 20 25 30 Asp Trp Ser Ile Asp IleLys Lys Tyr Gln Val Leu Val Gly Glu Pro 35 40 45 Val Arg Ile Lys Cys AlaLeu Phe Tyr Gly Tyr Ile Arg Thr Asn Tyr 50 55 60 Ser Leu Ala Gln Ser AlaGly Leu Ser Leu Met Trp Tyr Lys Ser Ser 65 70 75 80 Gly Pro Gly Asp PheGlu Glu Pro Ile Ala Phe Asp Gly Ser Arg Met 85 90 95 Ser Lys Glu Glu AspSer Ile Trp Phe Arg Pro Thr Leu Leu Gln Asp 100 105 110 Ser Gly Leu TyrAla Cys Val Ile Arg Asn Ser Thr Tyr Cys Met Lys 115 120 125 Val Ser IleSer Leu Thr Val Gly Glu Asn Asp Thr Gly Leu Cys Tyr 130 135 140 Asn SerLys Met Lys Tyr Phe Glu Lys Ala Glu Leu Ser Lys Ser Lys 145 150 155 160Glu Ile Ser Cys Arg Asp Ile Glu Asp Phe Leu Leu Pro Thr Arg Glu 165 170175 Pro Glu Ile Leu Trp Tyr Lys Glu Cys Arg Thr Lys Thr Trp Arg Pro 180185 190 Ser Ile Val Phe Lys Arg Asp Thr Leu Leu Ile Arg Glu Val Arg Glu195 200 205 Asp Asp Ile Gly Asn Tyr Thr Cys Glu Leu Lys Tyr Gly Gly PheVal 210 215 220 Val Arg Arg Thr Thr Glu Leu Thr Val Thr Ala Pro Leu ThrAsp Lys 225 230 235 240 Pro Pro Lys Leu Leu Tyr Pro Met Glu Ser Lys LeuThr Ile Gln Glu 245 250 255 Thr Gln Leu Gly Asp Ser Ala Asn Leu Thr CysArg Ala Phe Phe Gly 260 265 270 Tyr Ser Gly Asp Val Ser Pro Leu Ile TyrTrp Met Lys Gly Glu Lys 275 280 285 Phe Ile Glu Asp Leu Asp Glu Asn ArgVal Trp Glu Ser Asp Ile Arg 290 295 300 Ile Leu Lys Glu His Leu Gly GluGln Glu Val Ser Ile Ser Leu Ile 305 310 315 320 Val Asp Ser Val Glu GluGly Asp Leu Gly Asn Tyr Ser Cys Tyr Val 325 330 335 Glu Asn Gly Asn GlyArg Arg His Ala Ser Val Leu Leu His Lys Arg 340 345 350 Glu Leu Met TyrThr Val Glu Leu Ala Gly Gly Leu Gly Ala Ile Leu 355 360 365 Leu Leu LeuVal Cys Leu Val Thr Ile Tyr Lys Cys Tyr Lys Ile Glu 370 375 380 Ile MetLeu Phe Tyr Arg Asn His Phe Gly Ala Glu Glu Leu Asp Gly 385 390 395 400Asp Asn Lys Asp Tyr Asp Ala Tyr Leu Ser Tyr Thr Lys Val Asp Pro 405 410415 Asp Gln Trp Asn Gln Glu Thr Gly Glu Glu Glu Arg Phe Ala Leu Glu 420425 430 Ile Leu Pro Asp Met Leu Glu Lys His Tyr Gly Tyr Lys Leu Phe Ile435 440 445 Pro Asp Arg Asp Leu Ile Pro Thr Gly Thr Tyr Ile Glu Asp ValAla 450 455 460 Arg Cys Val Asp Gln Ser Lys Arg Leu Ile Ile Val Met ThrPro Asn 465 470 475 480 Tyr Val Val Arg Arg Gly Trp Ser Ile Phe Glu LeuGlu Thr Arg Leu 485 490 495 Arg Asn Met Leu Val Thr Gly Glu Ile Lys ValIle Leu Ile Glu Cys 500 505 510 Ser Glu Leu Arg Gly Ile Met Asn Tyr GlnGlu Val Glu Ala Leu Lys 515 520 525 His Thr Ile Lys Leu Leu Thr Val IleLys Trp His Gly Pro Lys Cys 530 535 540 Asn Lys Leu Asn Ser Lys Phe TrpLys Arg Leu Gln Tyr Glu Met Pro 545 550 555 560 Phe Lys Arg Ile Glu ProIle Thr His Glu Gln Ala Leu Asp Val Ser 565 570 575 Glu Gln Gly Pro PheGly Glu Leu Gln Thr Val Ser Ala Ile Ser Met 580 585 590 Ala Ala Ala ThrSer Thr Ala Leu Ala Thr Ala His Pro Asp Leu Arg 595 600 605 Ser Thr PheHis Asn Thr Tyr His Ser Gln Met Arg Gln Lys His Tyr 610 615 620 Tyr ArgSer Tyr Glu Tyr Asp Val Pro Pro Thr Gly Thr Leu Pro Leu 625 630 635 640Thr Ser Ile Gly Asn Gln His Thr Tyr Cys Asn Ile Pro Met Thr Leu 645 650655 Ile Asn Gly Gln Arg Pro Gln Thr Lys Ser Ser Arg Glu Gln Asn Pro 660665 670 Asp Glu Ala His Thr Asn Ser Ala Ile Leu Pro Leu Leu Pro Arg Glu675 680 685 Thr Ser Ile Ser Ser Val Ile Trp 690 695 5 657 DNA Homosapiens 5 atgtcctttg tgggggagaa ctcaggagtg aaaatgggct ctgaggactgggaaaaagat 60 gaaccccagt gctgcttaga agacccggct gtaagccccc tggaaccaggcccaagcctc 120 cccaccatga attttgttca cacaagtcca aaggtgaaga acttaaacccgaagaaattc 180 agcattcatg accaggatca caaagtactg gtcctggact ctgggaatctcatagcagtt 240 ccagataaaa actacatacg cccagagatc ttctttgcat tagcctcatccttgagctca 300 gcctctgcgg agaaaggaag tccgattctc ctgggggtct ctaaaggggagttttgtctc 360 tactgtgaca aggataaagg acaaagtcat ccatcccttc agctgaagaaggagaaactg 420 atgaagctgg ctgcccaaaa ggaatcagca cgccggccct tcatcttttatagggctcag 480 gtgggctcct ggaacatgct ggagtcggcg gctcaccccg gatggttcatctgcacctcc 540 tgcaattgta atgagcctgt tggggtgaca gataaatttg agaacaggaaacacattgaa 600 ttttcatttc aaccagtttg caaagctgaa atgagcccca gtgaggtcagcgattag 657 6 594 DNA Homo sapiens 6 atgtcctttg tgggggagaa ctcaggagtgaaaatgggct ctgaggactg ggaaaaagat 60 gaaccccagt gctgcttaga aggtccaaaggtgaagaact taaacccgaa gaaattcagc 120 attcatgacc aggatcacaa agtactggtcctggactctg ggaatctcat agcagttcca 180 gataaaaact acatacgccc agagatcttctttgcattag cctcatcctt gagctcagcc 240 tctgcggaga aaggaagtcc gattctcctgggggtctcta aaggggagtt ttgtctctac 300 tgtgacaagg ataaaggaca aagtcatccatcccttcagc tgaagaagga gaaactgatg 360 aagctggctg cccaaaagga atcagcacgccggcccttca tcttttatag ggctcaggtg 420 ggctcctgga acatgctgga gtcggcggctcaccccggat ggttcatctg cacctcctgc 480 aattgtaatg agcctgttgg ggtgacagataaatttgaga acaggaaaca cattgaattt 540 tcatttcaac cagtttgcaa agctgaaatgagccccagtg aggtcagcga ttag 594 7 474 DNA Homo sapiens 7 atgtcctttgtgggggagaa ctcaggagtg aaaatgggct ctgaggactg ggaaaaagat 60 gaaccccagtgctgcttaga agagatcttc tttgcattag cctcatcctt gagctcagcc 120 tctgcggagaaaggaagtcc gattctcctg ggggtctcta aaggggagtt ttgtctctac 180 tgtgacaaggataaaggaca aagtcatcca tcccttcagc tgaagaagga gaaactgatg 240 aagctggctgcccaaaagga atcagcacgc cggcccttca tcttttatag ggctcaggtg 300 ggctcctggaacatgctgga gtcggcggct caccccggat ggttcatctg cacctcctgc 360 aattgtaatgagcctgttgg ggtgacagat aaatttgaga acaggaaaca cattgaattt 420 tcatttcaaccagtttgcaa agctgaaatg agccccagtg aggtcagcga ttag 474 8 218 PRT Homosapiens 8 Met Ser Phe Val Gly Glu Asn Ser Gly Val Lys Met Gly Ser GluAsp 1 5 10 15 Trp Glu Lys Asp Glu Pro Gln Cys Cys Leu Glu Asp Pro AlaVal Ser 20 25 30 Pro Leu Glu Pro Gly Pro Ser Leu Pro Thr Met Asn Phe ValHis Thr 35 40 45 Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe Ser IleHis Asp 50 55 60 Gln Asp His Lys Val Leu Val Leu Asp Ser Gly Asn Leu IleAla Val 65 70 75 80 Pro Asp Lys Asn Tyr Ile Arg Pro Glu Ile Phe Phe AlaLeu Ala Ser 85 90 95 Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro IleLeu Leu Gly 100 105 110 Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp LysAsp Lys Gly Gln 115 120 125 Ser His Pro Ser Leu Gln Leu Lys Lys Glu LysLeu Met Lys Leu Ala 130 135 140 Ala Gln Lys Glu Ser Ala Arg Arg Pro PheIle Phe Tyr Arg Ala Gln 145 150 155 160 Val Gly Ser Trp Asn Met Leu GluSer Ala Ala His Pro Gly Trp Phe 165 170 175 Ile Cys Thr Ser Cys Asn CysAsn Glu Pro Val Gly Val Thr Asp Lys 180 185 190 Phe Glu Asn Arg Lys HisIle Glu Phe Ser Phe Gln Pro Val Cys Lys 195 200 205 Ala Glu Met Ser ProSer Glu Val Ser Asp 210 215 9 197 PRT Homo sapiens 9 Met Ser Phe Val GlyGlu Asn Ser Gly Val Lys Met Gly Ser Glu Asp 1 5 10 15 Trp Glu Lys AspGlu Pro Gln Cys Cys Leu Glu Gly Pro Lys Val Lys 20 25 30 Asn Leu Asn ProLys Lys Phe Ser Ile His Asp Gln Asp His Lys Val 35 40 45 Leu Val Leu AspSer Gly Asn Leu Ile Ala Val Pro Asp Lys Asn Tyr 50 55 60 Ile Arg Pro GluIle Phe Phe Ala Leu Ala Ser Ser Leu Ser Ser Ala 65 70 75 80 Ser Ala GluLys Gly Ser Pro Ile Leu Leu Gly Val Ser Lys Gly Glu 85 90 95 Phe Cys LeuTyr Cys Asp Lys Asp Lys Gly Gln Ser His Pro Ser Leu 100 105 110 Gln LeuLys Lys Glu Lys Leu Met Lys Leu Ala Ala Gln Lys Glu Ser 115 120 125 AlaArg Arg Pro Phe Ile Phe Tyr Arg Ala Gln Val Gly Ser Trp Asn 130 135 140Met Leu Glu Ser Ala Ala His Pro Gly Trp Phe Ile Cys Thr Ser Cys 145 150155 160 Asn Cys Asn Glu Pro Val Gly Val Thr Asp Lys Phe Glu Asn Arg Lys165 170 175 His Ile Glu Phe Ser Phe Gln Pro Val Cys Lys Ala Glu Met SerPro 180 185 190 Ser Glu Val Ser Asp 195 10 157 PRT Homo sapiens 10 MetSer Phe Val Gly Glu Asn Ser Gly Val Lys Met Gly Ser Glu Asp 1 5 10 15Trp Glu Lys Asp Glu Pro Gln Cys Cys Leu Glu Glu Ile Phe Phe Ala 20 25 30Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Glu Lys Gly Ser Pro Ile 35 40 45Leu Leu Gly Val Ser Lys Gly Glu Phe Cys Leu Tyr Cys Asp Lys Asp 50 55 60Lys Gly Gln Ser His Pro Ser Leu Gln Leu Lys Lys Glu Lys Leu Met 65 70 7580 Lys Leu Ala Ala Gln Lys Glu Ser Ala Arg Arg Pro Phe Ile Phe Tyr 85 9095 Arg Ala Gln Val Gly Ser Trp Asn Met Leu Glu Ser Ala Ala His Pro 100105 110 Gly Trp Phe Ile Cys Thr Ser Cys Asn Cys Asn Glu Pro Val Gly Val115 120 125 Thr Asp Lys Phe Glu Asn Arg Lys His Ile Glu Phe Ser Phe GlnPro 130 135 140 Val Cys Lys Ala Glu Met Ser Pro Ser Glu Val Ser Asp 145150 155 11 8 PRT Artificial sequence antigenic peptide used in fusionproteins 11 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 12 27 PRT Artificialsequence leucine zipper polypeptide 12 Pro Asp Val Ala Ser Leu Arg GlnGln Val Glu Ala Leu Gln Gly Gln 1 5 10 15 Val Gln His Leu Gln Ala AlaPhe Ser Gln Tyr 20 25 13 33 PRT Artificial sequence leucine zipperpolypeptide 13 Arg Met Lys Gln Ile Glu Asp Lys Ile Glu Glu Ile Leu SerLys Ile 1 5 10 15 Tyr His Ile Glu Asn Glu Ile Ala Arg Ile Lys Lys LeuIle Gly Glu 20 25 30 Arg 14 8 PRT Artificial sequence polymorphicsequence from exon 2 of Tango 77 14 Pro Ala Gly Ser Pro Leu Glu Pro 1 515 8 PRT Artificial sequence polymorphic sequence from exon 2 of Tango77 15 Pro Ala Val Ser Pro Leu Glu Pro 1 5

What is claimed is:
 1. An isolated polynucleotide comprising a nucleic acid molecule selected from the group consisting of: a) the polynucleotide of SEQ ID NO:1; b) the polynucleotide of SEQ ID NO:5; c) the polynucleotide of SEQ ID NO:6; d) the polynucleotide of SEQ ID NO:7; and, e) a polynucleotide that is capable of hybridizing to a polynucleotide of a)-d) under conditions of moderate stringency that include 50% formamide, 6×SSC at about 42° C., wherein the polypeptide encoded by the polynucleotide binds an IL-1R family member.
 2. An isolated polynucleotide comprising a nucleic acid molecule that encodes a polypeptide selected from the group consisting of: a) a polypeptide comprising SEQ ID NO:3; b) a polypeptide comprising SEQ ID NO:8; c) a polypeptide comprising SEQ ID NO:9; d) a polypeptide comprising SEQ ID NO:10; e) a polypeptide that is at least 80% identical to a polypeptide of a)-d), wherein the polypeptide binds an IL-1R family member; and, f) a fragment of the polypeptide of a)-e), wherein the fragment binds an IL-1R family member.
 3. An isolated polynucleotide comprising a nucleic acid molecule selected from the group consisting of: a) the polynucleotide nucleotide of SEQ ID NO:2; and b) a polynucleotide that encodes a polypeptide comprising SEQ ID NO:4; and, c) a polynucleotide encoding a polypeptide comprising a polypeptide that is at least 80% identical SEQ ID NO:4, wherein the polypeptide binds an IL-1 family member.
 4. An isolated polypeptide comprising a polypeptide selected from the group consisting of: a) a polypeptide comprising SEQ ID NO:3; b) a polypeptide comprising SEQ ID NO:8; c) a polypeptide comprising SEQ ID NO:9; d) a polypeptide comprising SEQ ID NO:10; e) a polypeptide that is at least 80% identical to a polypeptide of a)-d), wherein the polypeptide binds an IL-1R family member; and f) a fragment of a polypeptide of a)-e), wherein the fragment binds an IL-1R family member.
 5. An isolated polypeptide comprising a polypeptide selected from the group consisting of: a) the polypeptide of SEQ ID NO:4; b) a polypeptide that is at least 80% identical to a polypeptide of a), wherein the polypeptide binds an IL-1 family member; and c) a fragment of a polypeptide of a) or b), wherein the fragment binds an IL-1 family member.
 6. A vector comprising a polynucleotide of claim
 1. 7. A vector comprising a polynucleotide of claim
 2. 8. A vector comprising a polynucleotide of claim
 3. 9. A host cell transformed or transfected with an expression vector of claim
 6. 10. A host cell transformed or transfected with an expression vector of claim
 7. 11. A host cell transformed or transfected with an expression vector of claim
 8. 12. A method for preparing a polypeptide, the method comprising culturing a host cell of claim 9 under conditions promoting expression of the polypeptide.
 13. A method for preparing a polypeptide, the method comprising culturing a host cell of claim 10 under conditions promoting expression of the polypeptide.
 14. A method for preparing a polypeptide, the method comprising culturing a host cell of claim 11 under conditions promoting expression of the polypeptide.
 15. An oligomeric polypeptide comprising a polypeptide of claim
 4. 16. An oligomeric polypeptide comprising a polypeptide of claim
 5. 17. An antibody that binds a polypeptide of claim
 4. 18. An antibody that binds a polypeptide of claim
 5. 19. A method for screening a test compound to determine its affect on the ability of a IL-1 zeta polypeptide to increase or decrease IL-12 expression and or TNF-alpha expression, the method comprising: a) contacting a test compound and an IL-1 zeta polypeptide with cells capable of expressing IL-12 and/or TNF; and, b) analyzing the culture for IL-12 and/or TNF, wherein, if the IL-12 or TNF expression differs from the level of expression that is observed in the absence of test compound, the test compound affects IL-12 and/or TNF expression, and wherein, the IL-1 zeta polypeptide comprises a polypeptide selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, or fragment of the polypeptide of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10 and the fragment is capable of upregulating IL-12 or TNF expression.
 20. A method for increasing IL-12 production in an individual, the method comprising administering an IL-1 zeta polypeptide to the individual in an amount sufficient to increase IL-12 production, wherein the IL-1 zeta polypeptide comprises a polypeptide selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, or fragment of the polypeptide of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10 and the fragment is capable of upregulating IL-12 or TNF expression.
 21. A method for treating inflammation, the method comprising administering an antagonist of an IL-1 zeta polypeptide to an individual afflicted with an inflammatory condition, wherein the IL-1 zeta polypeptide comprises a polypeptide selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, or fragment of the polypeptide of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10 and the fragment is capable of upregulating IL-12 or TNF expression.
 22. A method for treating auto-immune disease, the method comprising administering an antagonist of an IL-1 zeta polypeptide to an individual afflicted with an auto-immune disease, wherein the IL-1 zeta polypeptide comprises a polypeptide selected from the group consisting of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, or fragment of the polypeptide of SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10 and the fragment is capable of upregulating IL-12 or TNF expression.
 23. The method of claim 22 wherein the auto-immune disease is selected from the group consisting of rheumatoid arthritis, SLE, myasthenia gravis, insulin-dependent diabetes mellitus, thyroiditis. 